CN102747020B - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN102747020B CN102747020B CN201210248300.2A CN201210248300A CN102747020B CN 102747020 B CN102747020 B CN 102747020B CN 201210248300 A CN201210248300 A CN 201210248300A CN 102747020 B CN102747020 B CN 102747020B
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Abstract
The invention discloses bacillus subtilis and an application thereof. The accession number of the bacillus subtilis BLG010 in the China General Microbiological Culture Collection Center is CGMCC No.5953. The bacillus subtilis BLG010 with number being CGMCC No.5953 can effectively inhibit the growth of fusarium oxysporum f. sp. cubense at low concentration and has great significance on control of banana wilt.
Description
Technical field
The present invention relates to a bacillus subtilis and application thereof.
Background technology
A kind of destructive disease causing infects in banana blight Shi You Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense), is the crushing soil-borne disease of banana production.Pathogenic bacteria is invaded from injured rhizome, by vascular bundle, to false stem and leaf portion, spreads, and stops up xylem vessel, until plant withered death.Banana blight bacteria is soil inhabitant, and its chlamydospore can survive 8～10 years in soil.
At present the control of blight is not also had to a kind of effective chemical pesticide, and life-time service chemical pesticide causes serious threat to environment and human health.Comparing with chemical prevention method, utilize Antagonistic Fungi controlling plant diseases to have the advantages such as environmentally friendly, cost is low, pathogenic bacteria is difficult for developing immunity to drugs, is the direction of following disease control.Although it has been found that a large amount of antagonistic bacteriums, current be separated to bacterium also far can not meet the requirement of Agriculture Production.Genus bacillus can produce heat-resisting, degeneration-resistant gemma, is conducive to production, the formulation of microbial inoculum and in environment, survives, surely grows and breeding.Both at home and abroad existing Biocontrol Bacillus is for preventing and treating the report of the leaf diseases such as the bacterium of the root diseases such as beans, wheat class, cotton and peanut and control vegetables, cherry, grape, cucurbit and English walnut and fungi.
Summary of the invention
A technical problem to be solved by this invention is to provide a bacillus subtilis.
Subtilis provided by the present invention is subtilis (Bacillus subtilis) BLG010, and registering on the books of Qi China Committee for Culture Collection of Microorganisms common micro-organisms center is numbered CGMCC No.5953.This bacterial classification has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on 04 06th, 2012.
It is opaque that subtilis of the present invention (Bacillus subtilis) BLG010 is creamy on nutrient agar medium (NA) substratum, and surface drying, has gauffer projection, and edge is irregular.Under microscope, single thalline is shaft-like, the blunt circle in two ends, and after gramstaining, even dyeing, and in visible cell, be dyed to bluish voilet gemma.
Another technical problem to be solved by this invention is to provide a kind of sterilant.
Sterilant provided by the present invention, its activeconstituents is above-mentioned subtilis (Bacillus subtilis) BLG010.
BLG010 specifically can be conidium or/and mycelium for described subtilis (Bacillus subtilis).
Described sterilant specifically can be used for the Plant diseases that control is caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).Described Plant diseases specifically can be banana blight.
Another technical problem to be solved by this invention is to provide a kind of method of preventing and treating banana blight.
The method of control banana blight provided by the present invention, comprises and adopts 1 * 10
8the step of the subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCC No.5953 bacteria suspension pouring banana seedlings base portion.
Experiment showed, with 1 * 10
8the subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCCNo.5953 bacteria suspension pouring banana seedlings base portion, reaches 81.4% to the protection effect of banana blight.Subtilis of the present invention (Bacillus subtilis) BLG010CGMCC No.5953 can effectively suppress the growth of fragrant Fusarium oxysporum under lower concentration, significant to the control of banana blight.
Strain name: subtilis
Latin name: Bacillus subtilis
Strain number: BLG010
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 04 06th, 2012
The preservation center numbering of registering on the books: CGMCC No.5953
Below in conjunction with specific embodiment, describe the present invention in detail, these embodiment are for understanding rather than restriction the present invention.
Accompanying drawing explanation
Fig. 1 is that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate is to the effect of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) Mycelial growth.
A is not for adding No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia on the PDA of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate.
B is for adding No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia on the PDA of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
NA nutrient solution in following embodiment: beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, is settled to 1000mL with distilled water; Its pH7.0.Percentage composition is wherein quality percentage composition.
NA substratum in following embodiment is the solid medium that adds agar to obtain in above-mentioned NA nutrient solution.
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
The Isolation and Identification of embodiment 1, subtilis (Bacillus subtilis) BLG010
1, the separation of subtilis (Bacillus subtilis) BLG010
From Chinese Hainan Province banana planting select blight to retransmit the fall ill banana plant of field of Tanaka, gather root soil sample.Soil sample is air-dry, after sieving, take 10g and put into the triangular flask that 90mL sterilized water and granulated glass sphere are housed, fully vibration, after standing 10min, rare to 1 * 10 by gradient concentration method
-3, 1 * 10
-4, 1 * 10
-5.Each gradient dilution liquid, in 80 ℃ of heating in water bath 20min, is got 100ul and is coated on nutrient agar medium (NA) isolation medium.30 ℃ of constant temperature culture 2d, according to representational single bacterium colony purifying of feature picking such as colony colour, form, humidity, transparency, edges, purifying is by gramstaining and spore staining, the Feature Selection bud pole bacterium such as thalline is shaft-like, product gemma proceed to NA slant medium and preserve, as bacterial strain to be measured.
Antagonism primary dcreening operation, sieve Antagonistic Fungi again: preparation Fusarium oxysporum Cuba No. 4 microspecies of specialized form (FOC4) spore liquid (approximately 2.0 * 10
5individual/mL), with 1/1000 ratio of substratum, draw spore suspension to PDA substratum and mix after to the large plate of square.Evenly bacterium is surveyed in some reception, cultivates 4 days at 28 ℃, observes and has or not inhibition zone.While sieving again, at the colony edge of cultivating the banana blight bacteria of 4 days, beat the bacterium piece that cut-off footpath is 5mm, moved to the dull and stereotyped central authorities of blank PDA.Then straight line inoculation is drawn for examination antagonistic bacterium by 5cm place in Ju center.2 strains testeds of every ware inoculation, only to inoculate indicator 28 ℃ of cultivation 6d in contrast.Continuous Observation records each Antagonistic Fungi colony length, width and antibacterial bandwidth, repeats 4 times, finally calculates inhibiting rate.Finally obtain subtilis (Bacillus subtilis) BLG010.
2, the evaluation of subtilis (Bacillus subtilis) BLG010
(1) Morphological Identification
The subtilis that separation and purification is arrived (Bacillus subtilis) BLG010 inoculation, on NA substratum, is cultivated 48h for 30 ℃, observes colonial morphology.Separately get the bacterium colony of cultivating 48h, suitably after dilution in micro-Microscopic observation.It is opaque that result shows that subtilis (Bacillus subtilis) BLG010 bacterial strain is creamy on NA substratum, and surface drying, has gauffer projection, and edge is irregular.Under microscope, single thalline is shaft-like, the blunt circle in two ends, and after gramstaining, even dyeing, and in visible cell, be dyed to bluish voilet gemma.
(2) Molecular Identification
The 16S rDNA complete sequence of subtilis (Bacillus subtilis) BLG010 is as shown in the sequence 1 in sequence table, and total length is 1429bp.
Subtilis (Bacillus subtilis) BLG010 has been preserved in registering on the books of Qi China Committee for Culture Collection of Microorganisms common micro-organisms center of China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) and has been numbered CGMCC No.5953 on 04 06th, 2012.
Embodiment 2, the antagonistic action of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 to No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia
1, subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate is to cause of disease mycelia restraining effect
In the PDA of molten state substratum, add respectively subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate (method according to following step 2 is prepared) to shake up system dull and stereotyped.A kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is front at the aseptic slide glass of culture dish central authorities to, pour substratum into and just do not have slide glass, diameter 5mm Fusarium oxysporum Cuba's No. 4 microspecies of specialized form (FOC4) bacterium piece is connected on slide glass, after 28 ℃ of cultivation 5d, slide glass is taken out and be directly placed in each mycelia form of processing of microscopy under Laser Scanning Confocal Microscope.Every processing repeats 3 times.Microscopic examination finds that the ferment filtrate of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 has stronger restraining effect to the growth of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia, mycelia is broken, and clears up (Fig. 1).Show that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate can obviously suppress the growth of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia.
2, the restraining effect of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 to No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) conidia germination
The restraining effect of the metabolite of this experiment employing subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluid test subtilis (Bacillus subtilis) BLG010CGMCC No.5953 to No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) conidia germination.
Subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluid is prepared as follows: subtilis (Bacillus subtilis) BLG010CGMCC No.5953 is inoculated into NA substratum, cultivate 2-3 days for 30 ℃, treat that it produces enough conidiums, with aseptic its spore of platinum loop scraping 2-3 ring, be inoculated in 250ml triangular flask (50ml NA nutrient solution is housed), put on temperature controllable shaking table, under 30 ℃ of conditions, 200rpm/min(rotation radius 13mm) constant-temperature shaking culture 36h; Then under aseptic condition according to 10%(volumn concentration) inoculum size access 500ml triangular flask in (200ml LB nutrient solution is housed); Postvaccinal shaking flask is under 31 ℃ of conditions, with 240rpm/min(rotation radius 13mm) speed oscillation cultivate 36 hours; Now bacterial strain has produced the bacteriostatic activity meta-bolites of high density in fermented liquid.Fermented liquid 5000rpm/min centrifugal 10-15min sedimentation mycelium and solid substance that above step is obtained, the aseptic filtering with microporous membrane of 0.22 μ m for supernatant liquor, collect filtrate (in this filtrate without subtilis (Bacillus subtilis) BLG010CGMCC No.5953), this filtrate is subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluid.
Get 600 μ l FOC4 spore liquid suspension (1 * 10
7cfu/mL), divide and be filled in 2ml centrifuge tube.In each centrifuge tube, add 200 μ l subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluids respectively again, mix and put 28 ℃ of constant temperature culture with pathogenic bacteria bacterium liquid respectively, take and add sterilized water as contrast.Every processing repeats 3 times, statistical computation antagonistic Bacillus 12h and the 24h inhibiting rate to Pathogen Causing Banana Fusarium Wilt conidia germination.
Result shows that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate was 89.25% at 12 hours to the inhibiting rate of FOC4 conidia germination, at 24 hours, to the inhibiting rate of FOC4 conidia germination, is 73.21%.
3, the restraining effect of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 to No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4)
Adopt dull and stereotyped face-off method, the antagonistic action of test subtilis (Bacillus subtilis) BLG010CGMCC No.5953 to No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4).Concrete grammar is as follows: at potato sucrose substratum, PDA (wherein adds 5g/L peptone, pH value is adjusted to 6.5) dull and stereotyped central No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) (pathogenic bacteria) the mycelia piece that diameter is 5mm that accesses, apart from 25mm place, pathogenic bacteria both sides, with transfering loop streak inoculation subtilis (Bacillus subtilis) BLG010CGMCC No.5953, the processing of Yi Zhijie Fusarium oxysporum Cuba No. 4 microspecies of specialized form (FOC4) mycelia piece in contrast, in 28 ℃ of incubators, cultivate 7 days, measure antibacterial bandwidth, every processing repeats 3 times.Result shows that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 is 11-13mm to the antibacterial bandwidth of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4).
Embodiment 3, the test of potted plant control banana blight
To grow 3 months, healthy Brazilian Banana transplantation of seedlings, to the sick soil flowerpot of (containing No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4)) is housed, is carried out 3 kinds of different processing: process one: when transplanting, by 200ml concentration, be 1 * 10
8the subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCC No.5953 bacterial strain bacteria suspension (obtaining with the spore of aqueous suspension subtilis (Bacillus subtilis) BLG010CGMCC No.5953) is along the pouring of banana base portion, 150 seedlings of co-processing; Process two: with 1 * 10
7the subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCC No.5953 bacteria suspension ((obtaining with the spore of aqueous suspension subtilis (Bacillus subtilis) BLG010CGMCC No.5953)) is along the pouring of banana base portion, 100 seedlings of co-processing; Process three for the contrast of pouring clear water, with isopyknic clear water, along banana base portion, water 150 seedlings of co-processing.In warmhouse booth, normal temperature is cultivated afterwards, occurs after disease symptom investigation disease index and protection effect until contrast:
Banana blight is divided into following 5 standards:
0 grade: plant is without withered and yellow symptom.
1 grade: slight withered and yellow symptom appears in plant lower blade, and tender leaf is intact, the slight brown stain of small part root system, there is water stain shape brown stain in stem.
2 grades: obvious withered and yellow symptom appears in plant lower blade, but tender leaf is intact, and brown stain appears in root system, and water stain shape brown stain appears in He Jia stem of stem.
3 grades: withered and yellow symptom appears in whole plant, root system brown stain is rotted, and in flakes, minority petiole occurs reddish brown the brown stain of He Jia stem of stem.
4 grades: withered death symptom appears in plant, the serious brown stain of root system is rotted.
Result is as shown in table 1, illustrates that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 has good protection effect to banana blight.
The protection effect of table 1. subtilis (Bacillus subtilis) BLG010CGMCC No.5953 to banana blight
| Process one | Process two | Process three (contrasts) | |
| Disease index | 16.3% | 41.3% | 87.4% |
| Protection effect | 81.4% | 52.7% | / |
Claims (7)
1. subtilis (Bacillus subtilis), it is characterized in that: the bacterial strain of described subtilis (Bacillus subtilis) number is BLG010, and registering on the books of Qi China Committee for Culture Collection of Microorganisms common micro-organisms center is numbered CGMCC No.5953.
2. a sterilant, its activeconstituents is subtilis claimed in claim 1 (Bacillus subtilis).
3. sterilant according to claim 2, is characterized in that: described subtilis (Bacillus subtilis) is for conidium is or/and mycelium.
4. according to the sterilant described in claim 2 or 3, it is characterized in that: the Plant diseases that described sterilant is caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense) for control.
5. sterilant according to claim 4, is characterized in that: described Plant diseases is banana blight.
6. the application of subtilis (Bacillus subtilis) BLG010CGMCC No.5953 in preparing bactericide; Described bactericide is used for preventing and treating banana blight; The target Jun Wei Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense) of described bactericide.
7. prevent and treat a method for banana blight, comprise and adopt 1 * 10
8the step of the subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCC No.5953 bacteria suspension pouring banana seedlings base portion.
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Application publication date: 20121024 Assignee: Hainan Bao Lvchun Agricultural Development Co., Ltd. Assignor: Chinese Academy of Tropical Agricultural Sciences Environment and Plant Protection Institute Contract record no.: 2016460000002 Denomination of invention: Bacillus subtilis and application thereof Granted publication date: 20140409 License type: Common License Record date: 20160321 |
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