CN105820986B - The special rhizosphere growth-promoting A Shi bacillus of one plant of blueberry and its application - Google Patents
The special rhizosphere growth-promoting A Shi bacillus of one plant of blueberry and its application Download PDFInfo
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Abstract
The invention discloses the special rhizosphere growth-promoting A Shi bacillus of one plant of blueberry and its application, the strain classification is named as A Shi bacillusBacillus aryabhattai, numbering is BPR078, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 7th, 2016, and deposit number is CGMCC No.11971.The blueberry rhizosphere growth-promoting A Shi bacillus of the present invention is made after microbial inoculum and is inoculated into blueberry cuttage seeding, can significantly accelerated blueberry take root, promote growth of cuttage seedlings, compared with the control, difference reaches the level of signifiance.Therefore, the present invention provides excellent strain resource for the special microbial manure of exploitation in future blueberry.
Description
Technical field
The present invention relates to the special rhizosphere growth-promoting A Shi bacillus of one plant of blueberry and its application.
Background technology
Blueberry (Vacciniumssp.L.) fruit color is gorgeous, unique flavor, contains substantial amounts of protein, fat, dimension life
The abundant nutritional ingredient such as element, pectin substance, SOD, flavones, potassium, phosphorus, calcium；Containing substantial amounts of natural pigment, and it has been investigated that
In various fruits, its pigment content highest, it has also become the raw material of health medicine.
Plant rhizosphere growth-promoting bacterium refers to existence in plant roots circle scope, has promotion to plant growth or has to pathogen
The beneficial bacteria of antagonism is referred to as, and has extremely important effect to plant growth and disease control.According to incompletely statistics, sent out
Now include the report that the plant rhizosphere growth-promoting bacterium of more than 20 category such as pseudomonad and bud pole bacterium has disease prevention growth-promoting potential.Plant
Thing rhizosphere growth-promoting endophytic bacteria can secrete plant growth-promoting material, including plant hormone, vitamin, amino acid and organic small point of other activity
Son and derivative etc.；The nutrient environment of plant rhizosphere can also be improved, effectively control plant disease etc..
The present inventor has filtered out blueberry rhizosphere growth-promoting bacillus fusiformis L13 from blueberry rhizosphere soil, and (preservation is compiled
Number it is CGMCC No.7068) and bacillus cereus (Bacillus cereus) L5 (deposit number is CGMCC No.7667),
The specific rhizosphere environment of blueberry is adapted to, and blueberry can be promoted to grow.
Blueberry is shallow root system, unrooted hair, is distributed in root system in thin solum, growing environment is more special, conventional work(more
Energy microbial inoculant is into blueberry soil, it is difficult to survives and colonizes, it is impossible to produce lasting, stable effect, based on above reason,
The PGPR strain resources of suitable blueberry are very deficient, continue abundant blueberry strain resource, the Tiny ecosystem ring to improving blueberry root system
Border, the growth of promotion blueberry, improvement blueberry quality are significant.
The content of the invention
It is an object of the invention to provide the special rhizosphere growth-promoting A Shi bacillus of one plant of blueberry and its application, the bacterial strain energy
The specific rhizosphere environment of blueberry is adapted to, and blueberry can be promoted to grow.
The present invention uses following technical scheme：
First purpose of the present invention is to provide one plant of A Shi bacillus, and the Classification And Nomenclature of the bacterial strain is A Shi gemma bars
Bacterium Bacillus aryabhattai, numbering is BPR078, is preserved in Chinese microorganism strain preservation on January 7th, 2016
Administration committee's common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, postcode 100101), deposit number is CGMCC No.11971.
Colony characteristicses and thalli morphology：
Colonial morphology is circular rule on LB culture mediums, and bacterium colony is larger, and culture 24h bacterium colony sizes are about 2-4.5mm,
Rat, is relatively moistened, slightly yellow, opaque, microscopy thalline is larger, shaft-like, there is gemma.
Physiological and biochemical property：
Citrate (Xi Mengshi) experiment is positive, and urease test is positive, and raffinose experiment is positive, and the experiment of L- rhamnoses is cloudy
Property, oxidizing ferment (Kovacs) negative, Fu-general negative, methyl red test feminine gender, indole test feminine gender, ONPG experiments
The positive, lysine decarboxylase negative, ornithine decarboxylase negative, dulcitol negative.
Function is qualitative and quantitative determines：
The bacterial strain of the present invention can produce protease, and the transparent loop diameter D of 48h generations is cultivated in casein substrate and is
16mm, colony diameter d are 6.1mm, and D/d is 2.62, and proteinase activity is 52.895 μ g/ml in quantitative determination zymotic fluid.
Using the type of U.S.'s Waters high performance liquid chromatographs 515, Waters2487 dual wavelength ultraviolets detector determines fermentation
Liquid kinds of organic acids and content.Chromatographic condition is chromatographic column：U.S. Thermo Hypersil GOLD 250 × 4.6mm of aQ,
5u；Mobile phase：10mmoLNH4H2PO4(phosphoric acid adjusts pH=2.3)：Methanol=98：2(V:V)；Detection wavelength：210nm, sensitivity：
0.5AUFS, sample size：10uL, flow velocity：0.8mL/min, column temperature：28℃.Testing result shows that BPR078 production concentration of oxalic acid is
452.36 μ g/ml, production acetic acid concentration is 9542 μ g/ml, and production citric acid concentration is 3865 μ g/ml.
Second object of the present invention is to provide a kind of microbial mixture, is characterized in the bacterium including at least the present invention
Strain, the microbial mixture can have comprising other promote plant growth (particularly promote blueberry growth) and with the present invention
Bacterial strain symbiosis microorganism.
Third object of the present invention is to provide a kind of product comprising bacterial strain of the present invention or microbial mixture of the present invention,
The product can be the forms such as microbial inoculum or composition.
It is preferred that, the product includes carrier；The carrier can be solid carrier or liquid-carrier.The solid carrier
Or liquid-carrier is conventional carrier material, wherein, solid carrier can be selected from clay, talcum, kaolin, montmorillonite, white
Carbon, zeolite, silica, corn flour, bean powder, polyvinyl alcohol and/or polyglycols；Liquid-carrier can be vegetable oil, mineral oil or water.
It is preferred that, the product be powder type, solution form, suspending agent form, tablet form, particle form or other
Form.
It is further preferred that the powder type can be freeze-dried powder form；The tablet form is to freeze containing described
The tablet form of dry powder and optional nutrient (such as vitamin, mineral salt).
The active component of the product be A Shi bacillus (Bacillus aryabhattai) BPR078, the activity into
Point can be with the living cells being cultured, the zymotic fluid of living cells, the filtrate of cell culture or cell and the mixture of filtrate
Form is present.
Fourth object of the present invention is the A Shi bacillus (Bacillus aryabhattai) of the protection present invention
BPR078 zymotic fluids.
The 5th purpose of the present invention is to provide a kind of bacterial strain of the invention, the microbial mixture or this hair of the present invention
Application of the bright product comprising bacterial strain of the present invention or microbial mixture of the present invention in plant growth is promoted, the plant tool
Body is blueberry.
The bacterial strain of the present invention can promote blueberry cuttage seedling rooting, promote blueberry growth, and can improve in blueberry rhizosphere soil
The activity of protease and invertase.
The 6th purpose of the present invention is to provide a kind of method for promoting blueberry to grow, and this method includes making what needs grew
The rhizosphere surrounding environment of blueberry is contacted with bacterial strain-A Shi bacillus (Bacillus aryabhattai) BPR078 of the present invention
The step of.
It is preferred that, the rhizosphere surrounding environment of the blueberry is blueberry rhizosphere soil.
It is preferred that, make the bacterial strain-A Shi bacillus of the rhizosphere surrounding environment and the present invention of the blueberry of needs growth
(Bacillus aryabhattai) is the step of BPR078 is contacted：With the bacterial strain-A Shi bacillus of the present invention
(Bacillus aryabhattai) BPR078 inoculations are needed in the blueberry rhizosphere soil of growth.
The beneficial effects of the invention are as follows：The blueberry rhizosphere growth-promoting A Shi bacillus of the present invention is inoculated into blueberry cuttage seeding,
Can significantly accelerated blueberry take root, promote growth of cuttage seedlings, and can improve the activity of protease and invertase in blueberry rhizosphere soil,
Compared with the control, difference reaches the level of signifiance.Therefore, the present invention provides excellent for the special microbial manure of exploitation in future blueberry
Good strain resource.
Brief description of the drawings
Fig. 1 is the colonial morphology figure of bacterial strain of the present invention.
Fig. 2 is the light microscope microscopy figure of bacterial strain of the present invention.
Fig. 3 is to contrast photo using the blueberry tissue culture seedling of the zymotic fluid of bacterial strain of the present invention and the blueberry tissue culture seedling of control group.
Embodiment
The A Shi bacillus of the present invention of embodiment 1 (Bacillus aryabhattai) BPR078 screening
(1) screening process
3, the collection blueberry rhizosphere soil sample in the different greenhouses in the good fertile blueberry planting base in Qingdao Jiangnan.Pass through gradient
Dilution method, isolates bacteria distribution thing from Rhizosphere sampling.The bacterium being separated to is purified by three zoning collimation methods, and led to
Cross microscopy and judge whether bacterial strain purifies, bacterium after purification is numbered, picking single bacterium colony is transferred on LB inclined-planes and preserves standby
With.
Bacterium, 28 DEG C, 180rpm, shaking table culture 4d are connect into the R2A fluid nutrient mediums containing L-Trp.Take 50 microlitres of bacterium
Suspension is dripped on whiteware plate, while adding 50 microlitres of Salkowski color solutions (50ml 35%HCLO4+1ml0.5mol/L
FeCl3).It regard the color solution for adding 50mg/L IAA as positive control.Whiteware plate is in sight after room temperature avoid light place 30min
Examine, the color person of reddening represent can producing IAA, just sift out the bacterium of producing IAA.
The bacterium of the producing IAA obtained to primary dcreening operation quantitative determines, and continues to screen the rhizosphere growth-promoting with application potential
Bacterium.Finally by the potted plant experiment of blueberry, determine that promotes the best bacterium of blueberry growth result, numbering is BPR078.
(2) colony characteristicses and thalli morphology
As depicted in figs. 1 and 2, colonial morphology is circular rule on LB culture mediums, and bacterium colony is larger, cultivates 24h bacterium colonies
Size is about 2-4.5mm, and rat is relatively moistened, slightly yellow, opaque, microscopy thalline is larger, shaft-like, there is gemma.
(3) physiological and biochemical property
Citrate (Xi Mengshi) experiment is positive, and urease test is positive, and raffinose experiment is positive, and the experiment of L- rhamnoses is cloudy
Property, oxidizing ferment (Kovacs) negative, Fu-general negative, methyl red test feminine gender, indole test feminine gender, ONPG experiments
The positive, lysine decarboxylase negative, ornithine decarboxylase negative, dulcitol negative.
(4) function is qualitative and quantitative determines
Bacterial strain can produce protease, and it is 16mm that 48h is cultivated in casein substrate and produces transparent loop diameter D, and bacterium colony is straight
Footpath d is 6.1mm, and D/d is 2.62, and proteinase activity is 52.895 μ g/ml in quantitative determination zymotic fluid.
Using the type of U.S.'s Waters high performance liquid chromatographs 515, Waters2487 dual wavelength ultraviolets detector determines fermentation
Liquid kinds of organic acids and content.Chromatographic condition is chromatographic column：U.S. Thermo Hypersil GOLD 250 × 4.6mm of aQ,
5u；Mobile phase：10mmoLNH4H2PO4(phosphoric acid adjusts pH=2.3)：Methanol=98：2(V:V)；Detection wavelength：210nm, sensitivity：
0.5AUFS, sample size：10uL, flow velocity：0.8mL/min, column temperature：28℃.Testing result shows that BPR078 production concentration of oxalic acid is
452.36 μ g/ml, production acetic acid concentration is 9542 μ g/ml, and production citric acid concentration is 3865 μ g/ml.
(5) 16S rDNA sequence analyses
The 16S rDNA gene orders of bacterial strain of the present invention, as shown in nucleotides sequence list.By surveyed 16S rDNA sequences with
Sequence alignment in GenBank databases.
Same branch is in A Shi bacillus (Bacillus aryabhattai) in BPR078 phylogenetic trees, its
The similitude of 16S rDNA sequences and A Shi bacillus (Bacillus aryabhattai) is up to 98.77%.With reference to bacterium colony shape
State, physiological and biochemical property and 16SrDNA sequence analyses, A Shi bacillus is accredited as by the strain BP R078 of the present invention
(Bacillus aryabhattai)。
Embodiment 2 has growth-promoting functions to blueberry growth
The BPR078 being stored on inclined-plane is activated in LB (peptone 10g with the method for three rides；NaCl 10g；Ferment
Female medicinal extract 5g；Agar 15-20g；Water 1000ml；PH 7.0-7.2, packing, 121 DEG C of sterilizing 20min) on flat board.Picking single bacterium colony
Into corresponding 10ml fluid nutrient mediums, 37 DEG C, 180rpm cultures 24h.Seed liquor is seeded to accordingly by 1% inoculum concentration
Fermented in 50ml fluid nutrient mediums, 37 DEG C, 180rpm cultures 2d arranges the Plug seedling experiment of blueberry tissue culture seedling.
Plug seedling is tested to be carried out in the fertile woods blueberry production base in Qingdao April 30 in 2015, from blueberry tissue culture seedling, each
Hole tray is 1 processing, 200 tissue-cultured seedling of cuttage.Each processing applies 100mL growth-promoting fermented liquids, is diluted to 1000mL uniform
Pour, normal management, July 30 in 2015 sampling survey, as a result as shown in table 1 below~3 and Fig. 3.As can be seen that inoculation BPR078
Blueberry cuttage seedling rooting can be promoted, promote blueberry growth, and the activity of protease and invertase in blueberry rhizosphere soil can be improved.
As can be seen here, BPR078 has very big application potential in blueberry cultivation kind.
The nutrition pot seedling of table 1 tests plant strain growth index
The nutrition pot seedling of table 2 tests biomass
The nutrition pot seedling of table 3 experiment soil enzymatic activities are determined
Embodiment 3
A kind of microbial mixture, is characterized in including the bacterial strain A Shi bacillus (Bacillus of the present invention
Aryabhattai) BPR078, also comprising other microorganisms, other described microorganisms for promote blueberry growth and with A Shi gemma
Bacillus (Bacillus aryabhattai) BPR078 does not produce the microorganism of antagonism.By experimental verification, the microorganism
Mixture is obviously promoted blueberry cuttage seedling rooting, promotes blueberry growth, and can improve protease and sucrose in blueberry rhizosphere soil
The activity of enzyme.
Embodiment 4
A kind of microbial inoculum comprising bacterial strain of the present invention or microbial mixture of the present invention, microbial inoculum includes carrier and the present invention
Described bacterial strain-A Shi bacillus (Bacillus aryabhattai) BPR078, the carrier can for solid carrier or
Liquid-carrier.The solid carrier or liquid-carrier are conventional carrier material, wherein, solid carrier can selected from clay,
Talcum, kaolin, montmorillonite, white carbon, zeolite, silica, corn flour, bean powder, polyvinyl alcohol and/or polyglycols；Liquid-carrier can
Think vegetable oil, mineral oil or water.The form of microbial inoculum is freeze-dried powder form.By experimental verification, the microbial inoculum is with A Shi gemma
Bacillus (Bacillus aryabhattai) BPR078 is active component, can promote blueberry cuttage seedling rooting, promotes blueberry life
It is long, and the activity of protease and invertase in blueberry rhizosphere soil can be improved.
Claims (9)
1. one plant of A Shi bacillus, it is characterized in that：The strain classification is named as A Shi bacillus（Bacillus aryabhattai）, numbering is BPR078, is preserved in Chinese microorganism strain preservation conservator on January 7th, 2016
Meeting common micro-organisms center, deposit number is CGMCC No.11971.
2. a kind of microbial mixture, it is characterized in that：Including at least the A Shi bacillus described in claim 1.
3. the product comprising the A Shi bacillus described in claim 1 or the microbial mixture described in claim 2.
4. product as claimed in claim 3, it is characterized in that：The product be powder type, solution form, suspending agent form,
Tablet form or particle form.
5. include the zymotic fluid of the A Shi bacillus described in claim 1.
6. described in the microbial mixture described in A Shi bacillus or claim 2 or claim 3 described in claim 1
Product or claim 5 described in zymotic fluid promote plant growth in application, it is characterized in that：The plant is blueberry.
7. application as claimed in claim 6, it is characterized in that：The A Shi bacillus being inoculated with described in claim 1 or right will
The fermentation liquid energy described in the product or claim 5 described in microbial mixture or the claim 3 described in 2 is asked to improve blueberry root
The activity of protease and invertase in the soil of border.
8. a kind of method for promoting blueberry to grow, it is characterized in that：This method includes ring around the rhizosphere for the blueberry for growing needs
The step of border is contacted with the A Shi bacillus described in claim 1.
9. method as claimed in claim 8, it is characterized in that, make the rhizosphere surrounding environment and claim of the blueberry of needs growth
The step of A Shi bacillus described in 1 contacts is：Being inoculated in using the A Shi bacillus described in claim 1 needs life
In the rhizosphere soil of long blueberry.
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| CN110468082B (en) * | 2019-09-19 | 2020-05-19 | 四川农业大学 | Bacillus aryabhattai OCB-6 and application thereof |
| CN110982743B (en) * | 2019-12-16 | 2022-03-15 | 沈阳农业大学 | Bacillus aryabhattai for promoting corn root development and application thereof |
| CN110999703B (en) * | 2019-12-20 | 2022-03-11 | 宜兴乾元黑色食品科技有限公司 | Method for improving secondary metabolite content of vaccinium bracteatum by infecting with megasporocyst mould |
| CN112410244B (en) * | 2020-09-30 | 2022-04-05 | 山东农业大学 | Bacillus aryabhattai and application thereof |
| CN113755382B (en) * | 2021-09-26 | 2023-08-11 | 山东农大肥业科技有限公司 | Bacillus aryabhattai NDFY-1 and application thereof |
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| CN105408468A (en) * | 2013-03-15 | 2016-03-16 | 斯波根生物技术公司 | Plant growth-promoting bacteria and methods of use |
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