CN102747020A - Bacillus subtilis and application thereof - Google Patents
Bacillus subtilis and application thereof Download PDFInfo
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- CN102747020A CN102747020A CN2012102483002A CN201210248300A CN102747020A CN 102747020 A CN102747020 A CN 102747020A CN 2012102483002 A CN2012102483002 A CN 2012102483002A CN 201210248300 A CN201210248300 A CN 201210248300A CN 102747020 A CN102747020 A CN 102747020A
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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
Banana blight is to infect a kind of destructive disease that causes by 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, spreads to false stem and leaf portion through vascular bundle, stops up xylem vessel, until the plant withered death.Banana blight bacteria is a soil inhabitant, and its chlamydospore can survive in soil 8～10 years.
Control to blight does not at present also have a kind of effective chemical agricultural chemicals, and the life-time service chemical pesticide causes serious threat to environment and human health.Comparing with the chemical prevention method, utilize antagonism bacterium controlling plant diseases to have advantages such as environmentally friendly, that 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, the present bacterium that is separated to also far can not be satisfied the requirement of agriculture prodn development.Genus bacillus can produce heat-resisting, degeneration-resistant gemma, and the production, the formulation processing that help microbial inoculum reach survival in environment, grow and breeding surely.Existing both at home and abroad biological and ecological methods to prevent plant disease, pests, and erosion genus bacillus is used to prevent and treat the report of leaf diseases such as bacterium and the fungi of root disease such as beans, wheat class, cotton and peanut and control vegetables, cherry, grape, cucurbit and English walnut.
Summary of the invention
A technical problem to be solved by this invention provides a bacillus subtilis.
Subtilis provided by the present invention is subtilis (Bacillus subtilis) BLG010, and it is numbered CGMCC No.5953 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.This bacterial classification has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 04 06th, 2012.
Subtilis of the present invention (Bacillus subtilis) BLG010 is creamy on nutrient agar medium (NA) substratum opaque, and surface drying has the gauffer projection, and the edge is irregular.The single thalline of microscopically is shaft-like, the blunt circle in two ends, and behind the gramstaining, even dyeing, and dyed the bluish voilet gemma in the visible cell.
Another technical problem to be solved by this invention provides a kind of sterilant.
Sterilant provided by the present invention, its activeconstituents are above-mentioned subtilis (Bacillus subtilis) BLG010.
BLG010 specifically can be conidium or/and mycelium for said subtilis (Bacillus subtilis).
Said sterilant specifically can be used for the Plant diseases that control is caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).Said Plant diseases specifically can be banana blight.
Another technical problem to be solved by this invention provides 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 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
Describe the present invention in detail below in conjunction with specific embodiment, these embodiment are used for understanding rather than restriction the present invention.
Description of drawings
Fig. 1 is that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate is to No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia restraining effect.
A is No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia on the PDA that does not add subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate.
B is No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia on the PDA that adds subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
NA nutrient solution among the following embodiment: beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0% is settled to 1000mL with zero(ppm) water; Its pH7.0.Percentage composition wherein is the quality percentage composition.
NA substratum among the following embodiment is in above-mentioned NA nutrient solution, to add the solid medium that agar obtains.
Following examples are used to explain the present invention, but are not used for limiting protection scope of the present invention.
Separation and the evaluation of embodiment 1, subtilis (Bacillus subtilis) BLG010
1, the separation of subtilis (Bacillus subtilis) BLG010
Select blight to retransmit the fall ill banana plant of field piece of Tanaka from banana planting ground, Chinese Hainan Province, gather the root soil sample.Air-dry soil sample, take by weighing 10g after sieving and put into the triangular flask that 90mL sterilized water and granulated glass sphere are housed, fully vibration, leave standstill 10min after, it is rare to 1 * 10 to press the gradient concentration method
-3, 1 * 10
-4, 1 * 10
-5Each gradient dilution liquid is got 100ul and is coated on nutrient agar medium (NA) isolation medium in 80 ℃ of heating in water bath 20min.30 ℃ of constant temperature culture 2d; According to representational single bacterium colony purifying of characteristic picking such as colony colour, form, humidity, transparency, edges; Purifying is after gramstaining and spore staining; Feature Selection bud pole bacterium such as thalline is shaft-like, product gemma change the NA slant medium over to and preserve, as bacterial strain to be measured.
The antagonism primary dcreening operation, sieve the antagonism bacterium again: preparation Fusarium oxysporum Cuba's No. 4 microspecies of specialized form (FOC4) spore liquid (about 2.0 * 10
5Individual/as mL), to draw behind spore suspension to the PDA substratum mixing to the big plate of square with 1/1000 ratio of substratum.Evenly bacterium is surveyed in some reception, cultivates 4 days down for 28 ℃, observes to have or not inhibition zone.When sieving again, beat cut-off at the colony edge of cultivating 4 days banana blight bacteria and directly be the bacterium piece of 5mm, it is moved to the dull and stereotyped central authorities of blank PDA.Then at 5cm place stroke straight line inoculation confession examination antagonistic bacterium apart from the center.2 strains testeds of every ware inoculation are cultivated 6d only to inoculate indicator for 28 ℃ as contrast.Each antagonism bacterium colony length of observed and recorded, width and antibacterial bandwidth repeat 4 times continuously, finally calculate inhibiting rate.Finally obtain subtilis (Bacillus subtilis) BLG010.
2, the evaluation of subtilis (Bacillus subtilis) BLG010
(1) morphology is identified
Subtilis (Bacillus subtilis) the BLG010 inoculation that separation and purification is arrived is cultivated 48h for 30 ℃ on the NA substratum, observe colonial morphology.Other gets the bacterium colony of cultivating 48h, and suitably the dilution back is observed in microscopically.It is opaque that the result shows that subtilis (Bacillus subtilis) BLG010 bacterial strain is creamy on the NA substratum, and surface drying has the gauffer projection, and the edge is irregular.The single thalline of microscopically is shaft-like, the blunt circle in two ends, and behind the gramstaining, even dyeing, and dyed the bluish voilet gemma in the visible cell.
(2) Molecular Identification
The 16S rDNA complete sequence of subtilis (Bacillus subtilis) BLG010 is shown in the sequence in the sequence table 1, and total length is 1429bp.
Subtilis (Bacillus subtilis) BLG010 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC), and it was numbered CGMCC No.5953 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center on 04 06th, 2012.
Embodiment 2, subtilis (Bacillus subtilis) BLG010CGMCC No.5953 are to the antagonistic action of 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 subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate (method according to following step 2 prepares) respectively and shake up the system flat board.A kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices is preceding at the aseptic slide glass of petridish 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 the slide glass, behind 28 ℃ of cultivation 5d slide glass is taken out the mycelia form that directly places each processing of microscopy under the Laser Scanning Confocal Microscope.Every processing repetition 3 times.The ferment filtrate of microscopic examination discovery 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, subtilis (Bacillus subtilis) BLG010CGMCC No.5953 is to the restraining effect of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) conidia germination
The restraining effect of the metabolite of 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 adopted in this experiment.
Subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluid prepares according to following method: BLG010CGMCC No.5953 is inoculated into the NA substratum with subtilis (Bacillus subtilis); Cultivated 2-3 days for 30 ℃; Treat that it produces the conidium of capacity; Scrape with aseptic platinum loop and to get its spore 2-3 ring and be inoculated in the 250ml triangular flask (50ml NA nutrient solution is housed); Put on the temperature controllable shaking table, under 30 ℃ of conditions, 200rpm/min (rotation radius 13mm) constant-temperature shaking culture 36h; Inoculum size according to 10% (volumn concentration) inserts in the 500ml triangular flask (200ml LB nutrient solution is housed) under aseptic condition then; The postvaccinal bottle that shakes was cultivated 36 hours with the speed oscillation of 240rpm/min (rotation radius 13mm) under 31 ℃ of conditions; This moment, bacterial strain produced the bacteriostatic activity meta-bolites of high density in fermented liquid.Fermented liquid 5000rpm/min centrifugal 10-15min sedimentation mycelium and solid substance with above step acquisition; Supernatant is with the aseptic filtering with microporous membrane of 0.22 μ m; Collect filtrating (not having subtilis (Bacillus subtilis) BLG010CGMCC No.5953 in this filtrating), this filtrating is subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluid.
Get 600 μ l FOC4 spore liquid suspension-s (1 * 10
7Cfu/mL), branch is filled in the 2ml centrifuge tube.In each centrifuge tube, adding 200 μ l subtilis (Bacillus subtilis) BLG010CGMCC No.5953 fermented supernatant fluids respectively again, put 28 ℃ of constant temperature culture with pathogenic bacteria bacterium liquid mixing respectively, is contrast to add sterilized water.Every processing repetition 3 times, statistical computation antagonism genus bacillus 12h and 24h are to the inhibiting rate of banana blight pathogenic bacteria conidia germination.
The result shows that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 ferment filtrate is 89.25% at 12 hours inhibiting rates to the FOC4 conidia germination, is 73.21% at 24 hours inhibiting rates to the FOC4 conidia germination.
3, subtilis (Bacillus subtilis) BLG010CGMCC No.5953 is to the restraining effect of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4)
Adopt dull and stereotyped face-off method, test subtilis (Bacillus subtilis) BLG010CGMCC No.5953 is to the antagonistic action of No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4).Concrete grammar is following: PDA (wherein adds the 5g/L peptone at the potato sucrose substratum; The pH value is adjusted to 6.5) to insert diameter be No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) (pathogenic bacteria) the mycelia piece of 5mm in dull and stereotyped central authorities; Apart from 25mm place, pathogenic bacteria both sides; With transfering loop streak inoculation subtilis (Bacillus subtilis) BLG010CGMCC No.5953, as contrast, in 28 ℃ of incubators, cultivated 7 days with the processing that only connects No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4) mycelia piece; Measure antibacterial bandwidth, every processing repetition 3 times.The 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 seedlings is transplanted to the sick soil flowerpot of (containing No. 4 microspecies of Fusarium oxysporum Cuba specialized form (FOC4)) is housed, and carries out 3 kinds of different processing: handle one: when transplanting, using 200ml concentration is 1 * 10
8The subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCC No.5953 bacterial strain bacteria suspension (spore with aqueous suspension subtilis (Bacillus subtilis) BLG010CGMCC No.5953 obtains) is along the pouring of banana base portion, 150 seedlings of co-processing; Handle two: with 1 * 10
7The subtilis of cfu/mL (Bacillus subtilis) BLG010CGMCC No.5953 bacteria suspension ((spore with aqueous suspension subtilis (Bacillus subtilis) BLG010CGMCC No.5953 obtains)) is along the pouring of banana base portion, 100 seedlings of co-processing; Handle three for the contrast of pouring clear water, water 150 seedlings of co-processing with isopyknic clear water along the banana base portion.Normal temperature is cultivated in warmhouse booth afterwards, treat that disease symptom appears in contrast after, investigation disease index and protection effect:
Banana blight is divided into following 5 standards:
0 grade: plant does not have withered and yellow symptom.
1 grade: slight withered and yellow symptom appears in the plant lower blade, and tender leaf is intact, few slight brown stain of part root system, and water stain shape brown stain appears in stem.
2 grades: tangible withered and yellow symptom appears in the plant lower blade, but tender leaf is intact, and brown stain appears in root system, and water stain shape brown stain appears in stem and false stem.
3 grades: withered and yellow symptom appears in whole plant, and the root system brown stain is rotted, stem and the brown stain of false stem in flakes, the minority petiole occurs reddish brown.
4 grades: the withered death symptom appears in plant, and the serious brown stain of root system is rotted.
The result is as shown in table 1, explains that subtilis (Bacillus subtilis) BLG010CGMCC No.5953 has good protection effect to banana blight.
Table 1. subtilis (Bacillus subtilis) BLG010CGMCC No.5953 is to the protection effect of banana blight
| Handle one | Handle two | Handle three (contrasts) | |
| Disease index | 16.3% | 41.3% | 87.4% |
| Protection effect | 81.4% | 52.7% | / |
Claims (10)
1. subtilis (Bacillus subtilis); It is characterized in that: the bacterial strain of said subtilis (Bacillus subtilis) number is BLG010, and it is numbered CGMCC No.5953 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. the application of the described subtilis of claim 1 (Bacillus subtilis) in producing conidium and/or mycelium.
3. sterilant, its activeconstituents is the described subtilis of claim 1 (Bacillus subtilis).
4. sterilant according to claim 3 is characterized in that: said subtilis (Bacillus subtilis) is that conidium is or/and mycelium.
5. according to claim 3 or 4 described sterilant, it is characterized in that: said sterilant is used for the Plant diseases that control is caused by Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).
6. sterilant according to claim 5 is characterized in that: said Plant diseases is a banana blight.
7. the application of the described subtilis of claim 1 in the preparation bactericide.
8. application according to claim 7 is characterized in that: the target bacterium of said bactericide is Fusarium oxysporum Cuba specialized form (Fusarium oxysporum f.sp.cubense).
9. according to claim 7 or 8 described application, it is characterized in that: said bactericide is used to prevent and treat banana blight.
10. a method of preventing and treating banana blight comprises and adopts 1 * 10
8The step of the subtilis of cfu/mL (Bacillus subtilis) BLG010 CGMCC No.5953 bacteria suspension pouring banana seedlings base portion.
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| CN104946574A (en) * | 2015-07-21 | 2015-09-30 | 中国热带农业科学院环境与植物保护研究所 | Bacillus subtilis Baisha2C for restraining plant pathogenic fungi |
| CN106172506A (en) * | 2016-07-03 | 2016-12-07 | 安建慧 | A kind of prevention or the method for the treatment of banana Panama disease |
| CN107312734A (en) * | 2017-08-21 | 2017-11-03 | 广东省农业科学院农业资源与环境研究所 | Banana blight Antagonistic Fungi and biological organic fertilizer and its preparation method and application |
| CN108402093A (en) * | 2018-04-28 | 2018-08-17 | 信阳农林学院 | A kind of prevention rice sheath blight disease microorganism and plant source compound pesticide |
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| CN103555613A (en) * | 2013-10-15 | 2014-02-05 | 广西科技大学 | Bacillus subtilis having antagonistic effect on Streptococcus agalactiae and application thereof |
| CN104946574A (en) * | 2015-07-21 | 2015-09-30 | 中国热带农业科学院环境与植物保护研究所 | Bacillus subtilis Baisha2C for restraining plant pathogenic fungi |
| CN104946574B (en) * | 2015-07-21 | 2020-04-14 | 中国热带农业科学院环境与植物保护研究所 | Bacillus subtilis Baisha2C for inhibiting plant pathogenic fungi |
| CN109068649A (en) * | 2016-04-24 | 2018-12-21 | 拜耳农作物科学股份公司 | Fluopyram and/or bacillus subtilis are used to prevent and treat the purposes of the wilt disease of Musaceae plant |
| CN106172506A (en) * | 2016-07-03 | 2016-12-07 | 安建慧 | A kind of prevention or the method for the treatment of banana Panama disease |
| CN107312734B (en) * | 2017-08-21 | 2020-03-17 | 广东省农业科学院农业资源与环境研究所 | Banana wilt antagonistic bacteria and bio-organic fertilizer as well as preparation method and application thereof |
| CN107312734A (en) * | 2017-08-21 | 2017-11-03 | 广东省农业科学院农业资源与环境研究所 | Banana blight Antagonistic Fungi and biological organic fertilizer and its preparation method and application |
| CN108402093A (en) * | 2018-04-28 | 2018-08-17 | 信阳农林学院 | A kind of prevention rice sheath blight disease microorganism and plant source compound pesticide |
| CN112006036A (en) * | 2020-09-08 | 2020-12-01 | 仲恺农业工程学院 | Microbial suppository for disease resistance and yield increase of bananas as well as preparation method and application of microbial suppository |
| CN113966747A (en) * | 2021-06-30 | 2022-01-25 | 中国热带农业科学院环境与植物保护研究所 | Nano-silver composite bacillus agent for preventing and treating syzygium samarangense fruit rot and anthracnose of syzygium samarangense fruit and preparation method thereof |
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