CN105733982B - Bacillus amyloliquefaciens, microbial inoculum and preparation method thereof for preventing and treating two born of the same parents' branch-rot of blueberry hair color - Google Patents
Bacillus amyloliquefaciens, microbial inoculum and preparation method thereof for preventing and treating two born of the same parents' branch-rot of blueberry hair color Download PDFInfo
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Abstract
The present invention relates to a bacillus amyloliquefaciens Bacillus amyloliquefaciensHMQAU140045, on December 1st, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in Microbe Inst., Chinese Academy of Sciences, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number: CGMCC No.11768.The present invention provides an efficient microorganism for two born of the same parents' branch-rot of blueberry hair color, opens a new approach；Bacillus amyloliquefaciens strain HMQAU140045 of the present invention has significant preventive effect to two born of the same parents' branch-rot of blueberry hair color, and also has preferable preventive effect to blueberry branch-rot caused by other pathogens.
Description
Technical field
The invention belongs to technical field of plant disease biological control, and in particular to one plant of two born of the same parents' branch-rot of prevention and treatment blueberry hair color
Bacillus amyloliquefaciens HMQAU140045 screening, identification, two born of the same parents' branch-rot of fermentation, Antibacterial Activity and blueberry hair color
Prevention and treatment, belong to agricultural biological technical field.
Background technique
Blueberry belongs to Ericaceae Ericaceae, Vaccinium Vaccinium, is a kind of with the small of high economic value
Berry fruit tree is classified as one of big healthy food of the mankind five by FAO (Food and Agriculture Organization of the United Nation) because of its healthy nutritive value outstanding.It is blue
The certain kind of berries has become a nationwide emerging leading industry, and one is transformed agricultural production, and drives farmer richness, has the modern times
Agricultural features, and be the health industry of Chinese nation's health service, blueberry plantation industry also has become China's planting fruit trees industry
One of most popular industry.In recent years, blueberry cultivated area also expands rapidly therewith, national total area reaches cultivation within 2015
31210 hectares, 15300 hectares than 2013 have been increased sharply 51%.
However, with cultivated area expand rapidly with the time limit in plantation increase, the time limit increase, technical experience lack with
And the reasons such as mismanagement of individual casual household, the type of blueberry disease increases increasingly and the extent of injury is on the rise, and blueberry branch is withered
It is sick to have become a kind of Major Diseases for restricting blueberry yield and quality at present, and the cause of disease type of branch-rot is caused to be also multiple
Miscellaneous multiplicity, at present main pathogenic bacteria blueberry Phomopsis Phomopsis vaccinii of China Report, rodlike quasi- disk crinosity
Pestalotiopsis clavispora, grape seat chamber bacterium Botryosphaeria dothidea etc..In recent years, in Shandong Province blueness
A kind of new two born of the same parents Lasiodiplodia of branch-rot disease fungus vacation cocoa hair color is found in the blueberry disease survey of island city Jiangnan
Pseudotheobromae, the disease infection rate is rapider, constitutes threat to blueberry production.And the above branch-rot disease
Opportunistic pathogen can individually infect, also can Combined Infection.Discovery is investigated for the prevention and treatment of blueberry branch-rot, field is main at present or uses
Agro-chemicals control means, but food safety, pathogen drug resistance and ecological disruption caused by chemical agent is used for a long time and also must
It will become and produce upper problems faced.Therefore the new safely and effectively measure of one kind is found to prevent and treat two born of the same parents' branch-rot of blueberry hair color
It is the task of top priority.
Biological control is as environmentally friendly, and preventive effect is stable, persistently, is not likely to produce the measure of drug resistance, will push agriculture
The sustainable development of industry.Nowadays, research hotspot both domestic and external is had become for the research of biological control, using biocontrol bacteria come
Controlling plant diseases are a kind of main means of biological control.Through retrieving, inventor is in existing patent and non-patent literature
Relevant report and patent of the bacillus amyloliquefaciens for the two born of the same parents' branch-rot biological control of blueberry hair color are not retrieved.
Summary of the invention
It is an object of that present invention to provide the Bacillus amyloliquefaciens strain of prevention and treatment two born of the same parents' branch-rot of blueberry hair color, bacterial strain tools
There is the advantages of efficient, wide sterilization spectrum.
It is described to realize that technical scheme is as follows.
One bacillus amyloliquefaciens Bacillus amyloliquefaciensHMQAU140045, on December 1st, 2015
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in Microbe Inst., Chinese Academy of Sciences, preservation
Location: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number: CGMCC No.11768.
Using above-mentioned bacillus amyloliquefaciens HMQAU140045 produce three kinds of microbial bacterial agents (A, B, C), A activity at
It is divided into HMQAU140045 thallus, B activity ingredient is ferment filtrate, and C active constituent is thallus and ferment filtrate.
One aspect of the present invention, the microbial inoculum comprising bacillus amyloliquefaciens, the microbial inoculum can be those skilled in the art
Any microbial inoculum of any technological means preparation known to member.Such as the concentrating agents by liquid form after fermented and cultured, it is also possible to
The dry microbial inoculum obtained by freeze-drying.
A and C viable bacteria body number in mentioned microorganism microbial inoculum should be greater than 3.2x1010cfu/mL。
The preparation step of mentioned microorganism microbial inoculum is as follows:
(1) actication of culture, by the bacterial strain HMQAU140045 of low-temperature preservation in 28 DEG C of cultures in LB solid medium tablets
48h, then be transferred in LB solid slope culture medium and cultivate for 24 hours for 28 DEG C, the strain activated；
(2) seed liquor prepares the strain of one collarium step (1) of aseptic inoculation activation in 50mL LB liquid medium
130rpm, 30 DEG C of oscillations for 24 hours, collect thallus, and be transferred to 108Cfu/mL1 prepares seed liquor；
(3) seed liquor that step (2) obtains 1.5% is inoculated in Optimal compositions of fermentation medium in 28 by volume by fermented and cultured
DEG C, after 180r/min shakes training 84h, carry out microbial inoculum preparation.The preparation of A microbial inoculum, after shaking the fermentation liquid centrifugation that training obtains, thallus is with sterile
Twice, centrifugation, the thallus for scraping precipitating is placed in sterile water water washing, is configured to microbial inoculum A；B microbial inoculum preparation, fermentation liquid in 4 DEG C,
12000rpm is centrifuged 20 minutes acquisition supernatants, and by 0.22 μm of filter, collecting filtering liquid is microbial inoculum B；C microbial inoculum system
Standby, after fermentation liquid centrifugation, the thallus for scraping precipitating is placed in bacteria-free filtrate (not being diluted), is configured to microbial inoculum C.
Application of the above-mentioned bacillus amyloliquefaciens in two born of the same parents' branch-rot of blueberry hair color.
A in above-mentioned gained microbial bacterial agent is that cell concentration is diluted with water by the application method of microbial bacterial agent of the present invention
To 1 × 108Cfu/mL, B are ferment filtrate, and C is that cell concentration is diluted with ferment filtrate to 1 × 108Cfu/mL is withered in blueberry branch
It is applied before the onset of sick.
The screening of HMQAU140045 bacterial strain separates: HMQAU140045 bacterial strain is inventor from the Shandong Province Huangdao District Qing Shi
It is isolated in blueberry rhizosphere soil.In April, 2014 blueberry garden of the inventor from Qingdao of Shandong province Huangdao District, five point sampling methods
Rhizosphere Soil is acquired, 10g soil is weighed after mixing and is added in 250 triangular flasks, 90mL sterile water is added, 20min is vibrated on shaking table, it is quiet
It sets about 30 seconds, supernatant is 10-1Dilution, then it is diluted to 10 step by step-2, 10-3, 10-4, 10-5Equal extension rates, three after taking
The dilution 1mL of concentration and LB culture medium 9mL cooling after fusing is mixed into plate, and each concentration in triplicate, is trained at 28 DEG C
1-3d is supported, carries out the separation and purifying of bacterium, and using two born of the same parents' branch-rot bacterium of blueberry hair color as target, pass through tablet face-off method, spore
Sub- sprouting method and isolated shoot test carry out the screening of antagonistic strain, and final one plant of acquisition has two born of the same parents' branch-rot of blueberry hair color
The bacterial strain of obvious control efficiency, is named as HMQAU140045.
The taxonomic identification of 140045 microbial inoculum of HMQAU:
(1) identification by morphological characters
30 DEG C of culture 2d, 0.5~2.5mm of colony diameter, bacterium colony projection are creamy white, are impermeable on LB solid medium
Bright, round, edge is irregular, there is fold on surface, has crystalline material in colony edge.Thallus Gram-positive, it is rod-shaped,
(0.93 ± 0.24) μm~(2.01 ± 0.52) μm, single or arrangement in pairs, produce gemma.The physiological and biochemical test bacterium as the result is shown
Strain is facultative anaerobic bacteria, can hydrolyze starch, reduction nitrate, can utilize citrate, glucose, fructose, sucrose, sweet dew
Alcohol, catalase, oxidizing ferment and V-P reaction are the positive, and denitrification and clark and Lubsreaction are feminine gender.The bacterial strain is in 2%~7%NaCl
With under the acid condition of pH5 have tolerance, under the alkaline condition of 10%NaCl and pH9 do not have tolerance；4 DEG C cannot give birth to
Long, 51 DEG C can grow.Identify that the Preliminary Identification bacterium is bacillus amyloliquefaciens Bacillus by morphology and Physiology and biochemistry
amyloliquefaciens；
(2) Molecular Identification
Using HMQAU140045 genomic DNA as template, 16srDNA segment is expanded using primer 2 7f/1492r.
The primer sequence is 27f (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5 '-TACGGTTACCT
TGTTACGACTT-3').Amplification reaction system: 10 × Buffer, 2.5 μ L, 5mM dNTP 2 μ L, 10 μM of primer 2 7f and primer
Each 1 μ L of 1492r, Taq enzyme (5U/ μ L) 0.5 μ L, 2.5 μ L of template DNA, moisturizing to 25 μ L.Reaction condition: 94 DEG C of initial denaturations
5min；94 DEG C of denaturation 1min, 53 DEG C of annealing 1min, 72 DEG C of extension 1.5min, 30 recycle；Last 72 DEG C of extensions 10min.Amplification
Product carries out purifying and bidirectional sequencing after the detection of 1% agarose gel electrophoresis, by Sangon Biotech (Shanghai) Co., Ltd.,
Sequencing result exported after Sequencher5.0 software automatic assembling contig (Contig) and NCBI (http: //
Www.ncbi.nlm.gov GenBank is submitted to after) carrying out BLAST analysis in database.It is suitable to choose from GenBank again
Sequence carries out alignment through CLUSTALW2.0 software, and uses adjacent method (neighbor-with 5.05 software of MEGA
Joining analysis, NJ) phylogenetic tree construction, the number of repetition that wherein Bootstrap is examined is 1,000 time.As a result
Show that (GenBank is stepped on the bacterial strain with the bacillus amyloliquefaciens Bacillus amyloliquefaciens from soil in Yunnan
Record number reaches 99% for the homology of JN99860).Phylogenetic Analysis the result shows that, the bacterial strain and 4 plants of Bacillus
The sequence of amyloliquefaciens bacterial strain is located at the same branch of phylogenetic tree.Sequence analysis data and systematic growth
It is Bacillus amyloliquefaciens that tree position, which further demonstrates bacterial strain HMQAU140045,.
In summary morphological feature and 16SrDNA sequence homology compare analysis result and know that HMQAU140045 belongs to solution
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and it is different with existing Bacillus amyloliquefaciens strain,
It is a new strains.
The identification method of above-mentioned bacillus amyloliquefaciens strain HMQAU140045, including being with the genomic DNA of strain to be tested
Template, using 27f and 1492r as the amplification of primer, if gained PCR product is nucleotide sequence shown in SEQIDNo:1, as
HMQAU140045 bacterial strain.
The detection of the B microbial inoculum and C microbial inoculum of above-mentioned Bacillus amyloliquefaciens strain HMQAU140045, with protease and fiber
The detection culture medium detection of plain enzyme, if there is positive reaction, the as B microbial inoculum and C microbial inoculum of HMQAU140045 bacterial strain.
The advantages of the present invention: (1) present invention provides one efficiently for two born of the same parents' branch-rot of blueberry hair color
Microorganism opens a new approach；(2) Bacillus amyloliquefaciens strain HMQAU140045 of the present invention is to blueberry hair color two
Born of the same parents' branch-rot has significant preventive effect, and also there is preferable preventive effect to implement blueberry branch-rot caused by other pathogens.(3) originally
Invention microbial bacterial agent is to people, animal safety；It is not likely to produce drug resistance, reduces resistance risk；It, can reduction to be environmentally friendly
The application of fungicide is learned, the destruction to environment and the influence to human health are reduced, to realize the safe and efficient of blueberry production
Sustainable development.(4) low production cost of the invention, preparation method and application method are simple.
Detailed description of the invention
The present invention has following attached drawing:
Fig. 1 is antagonistic effect of the Bacillus amyloliquefaciens strain HMQAU140045 to two born of the same parents branch-rot bacterium of blueberry hair color；
Fig. 2 is that Bacillus amyloliquefaciens strain HMQAU140045 ferment filtrate sprouts false two born of the same parents conidium of cocoa hair color
The influence of hair；
Fig. 3 is Bacillus amyloliquefaciens strain HMQAU140045 colonial morphology and Gram's staining；
Fig. 4 be the antagonistic strain Bacillus amyloliquefaciens strain HMQAU140045 that construct based on 16SrDNA gene order with
NJ (neighbor-joining analysis) phylogenetic tree of Related Bacteria；
Fig. 5 is the protease and cellulase testing result of Bacillus amyloliquefaciens strain HMQAU140045.
Specific embodiment
Case study on implementation is for being explained further the present invention below, but is not construed as limiting the invention in any way.
Test method in following case study on implementation, unless otherwise specified, generally conventional method；Percentage in following case study on implementation contains
Amount, is weight percentage unless otherwise specified.
Embodiment 1
The separation and screening of antagonistic strain.
(1) separate: in April, 2014 acquires the Rhizosphere Soil of blueberry plant in the blueberry garden of Qingdao of Shandong province Huangdao District.
Using dilution-plate method isolated bacterial strain, culture medium is LB solid medium, and after growing bacterium colony, picking bacterium single bacterium is fallen within
The scribing line of LB solid plate purifies and is stored in mycology research department, Qingdao Agricultural University.
(2) primary dcreening operation: using false two born of the same parents of cocoa hair color as target, using face-off flat band method, the streak inoculation of PDA plate center is thin
Bacterium, the both ends away from bacterium 2.5cm are inoculated with pathogen bacteria cake, 25 DEG C of constant temperature incubations, and control is only inoculated with pathogen bacteria cake.Each processing
If 3 repetitions when covering with 3/4 culture dish wait compare, measure antibacterial bandwidth, filter out have to false two born of the same parents of cocoa hair color it is obvious short of money
The bacterium bacterial strain of anti-effect.
(3) secondary screening: the bacterium bacterial strain after primary dcreening operation prepares seed liquor, and 1mL seed liquor is taken to be inoculated in 50mL LB culture medium,
130r/min, 30 DEG C of constant-temperature shaking culture 48h.Fermentation liquid is centrifuged, 0.22 μm of filtering with microporous membrane prepares the sterile of Antagonistic Fungi
Filtrate.Using mycelial growth rate method, mixed for cooling (45~50 DEG C) of PDA culture medium by fermentation liquid bacteria-free filtrate and after melting
Even, inverted plate, the PDA culture medium isometric LB liquid medium is added is inoculated with diameter in plate center as control as 5mm
Two born of the same parents' germ bacteria cake of false cocoa hair color.Each processing sets 3 repetitions, and when covering with 3/4 culture dish wait compare, crossing method is surveyed
Control group and processing group colony diameter are measured, bacteriostasis rate is calculated, filters out the bacterium optimal to false two born of the same parents' antagonistic effect of cocoa hair color
Bacterial strain.
Inhibiting rate (%)=(control bacterium colony half (mm)-processing colony radius (mm))/control colony radius (mm) ×
100%
Test simultaneously using the measurement of both the above method screen the best antagonistic strain of obtained fungistatic effect to other three
Strain blueberry branch-rot bacteria stick shape intends disk crinosity Pestalotiopsis clavispora, grape seat chamber bacterium Botryosphaeria
The inhibiting effect of dothidea, blueberry Phomopsis Phomopsis vaccinii.
(4) result and analysis: using dilution-plate method from the Rhizosphere Soil of healthy blueberry plant isolated 20 plants of bacteriums,
Face-off flat band method filters out 7 plants of antagonistic strains for having obvious inhibitory effect to false two born of the same parents of cocoa hair color, accounts for separated bacterial strain
35%, wherein bacterial strain HMQAU140045 antagonistic effect is best, and antibacterial bandwidth reaches 14.50mm (Fig. 1-A).Choose the bacterial strain
LB ferment filtrate 10 times of progress mycelial growth rate method tests are diluted in PDA culture medium, the results showed that the fermentation of the bacterial strain
The inhibiting rate that filtrate grows false two born of the same parents mycelia of cocoa hair color can reach 100% (Fig. 1-B).In addition, HMQAU140045 pairs of bacterial strain
The antibacterial bandwidth of grape seat chamber bacterium, blueberry Phomopsis and rodlike quasi- disk stey is respectively 12.63mm, 18.3mm,
8.75mm.The bacterial strain dilutes 10 times of LB ferment filtrate to grape seat chamber bacterium, blueberry Phomopsis and rodlike quasi- disk stey
Inhibiting rate be respectively 100%, 89.67%, 76.74%.Illustrate bacterial strain HMQAU140045 to a variety of blueberry branch-rot pathogens
With preferable fungistatic effect.
Embodiment 2
The optimization of antagonistic strain HMQAU140045 fermentation medium and condition.
(1) optimization of fermentation medium
Using single factor test and the optimization method of orthogonal test, first passes through single factor experiment (carbon source, nitrogen source, inorganic salts) and determine
The appropriate value of optimal nutrient media components, reselection each component is horizontal, carries out orthogonal test.Test is with false two born of the same parents of cocoa hair color
For for trying pathogen.30 DEG C after access seed liquor, 130r/min shakes training 48h., with spectrophotometric determination difference factor pair
The influence of HMQAU140045 thallus yield.Using mycelial growth rate method measurement ferment filtrate to false two born of the same parents' of cocoa hair color
Bacteriostasis rate (carbon and nitrogen sources ferment filtrate dilutes 50 times, other 100 times of dilutions).
Single factor test optimization.
Carbon source optimizing: with glucose, sucrose, maltose, fructose, D- xylose, lactose, soluble starch, potato starch,
Corn flour is that 2% carbon source is added in basal medium (LB culture medium) for trying carbon source, and each carbon source sets three repetitions, with
The basal medium of carbon source is not added as control.
Nitrogen source optimization: with ammonium sulfate, urea, soya-bean cake, peanut cake, soybean powder, beef extract, peptone, tryptone, yeast
Powder.Optimal carbon source to be added in basal medium (LB culture medium) as control (CK), respectively with 2.0% nitrogen source equivalent substitution pair
According to the tryptone and yeast extract in culture medium, 3 repetitions of each processing.
Inorganic salts optimization: with CuSO4、MnSO4、MgSO4、CaCO3、K2HPO4To be added with most suitable carbon nitrogen source for trying inorganic salts
NaCl is control, respectively with the NaCl in 0.5% inorganic salts equivalent replacement control medium.
Orthogonal optimization: the optimum carbon source that is filtered out using single factor experiment, nitrogen source, inorganic salts select 4 factors as variance factor
3 horizontal L9(34) orthogonal arrage progress medium optimization test, determine the optimum proportioning (table 1) of culture medium each component.
(2) optimization of fermentation condition
The optimization of fermentation condition is divided into single factor experiment (revolving speed, temperature, initial pH, incubation time, inoculum concentration and dress liquid
Amount) and orthogonal test, on the basis of single factor experiment, the suitable horizontal progress orthogonal test of selection determines most preferably fermentation item
Part combination.Test is for trying pathogen with false two born of the same parents of cocoa hair color.
1 fermentation medium compositional factors water-glass of table.
Single factor experiment: under conditions of optimization obtains Optimal compositions of fermentation medium, pH (5.0,6.0,7.0,8.0 and are determined
9.0), cultivation temperature (24,28,32,36 and 40 DEG C), revolving speed (100,120,140,160,180 and 200rpm), liquid amount (20,
30,40,50 and 60mL), influence of the initial inoculum (1,2,4,6 and 8%) to bacteriostatic activity, each processing repeats three times.It surveys
Determine method with fermentation medium optimization (ferment filtrate dilutes 100 times).
Orthogonal test: using Optimal Medium, under conditions of most suitable revolving speed, when with cultivation temperature, initial pH, culture
Between, inoculum concentration and liquid amount be investigation factor, by orthogonal array L25(55) design 5 factors, 5 horizontal orthogonal tests
(table 2), influence of the more various fermentation condition combinations to the bacteria-free filtrate antibacterial activity of biocontrol bacterial strain fermentation liquid, determines most suitable hair
Ferment condition.Measuring method optimizes (ferment filtrate dilutes 100 times) with fermentation medium.
2 fermentation condition factor level table of table.
(1) optimum results of fermentation medium.
Single factor test optimization.
The single factor test the selection result of carbon source shows that when corn flour is as carbon source, bacterium amount is maximum after fermentation, and bacterium amount is big thereafter
Carbon source is not added in the small maltose > potato starch > lactose > soluble starch > D- xylose > glucose > sucrose > fructose > that is followed successively by；Suppression
The size of bacterium rate be followed successively by maltose > corn flour > potato starch > soluble starch > lactose > D- xylose > sucrose > glucose >
Fructose > carbon source is not added.When discovery is using corn flour as carbon source simultaneously, although bacteriostasis rate is not as good as maltose, there was no significant difference,
And corn flour as carbon source when bacterium amount highest, and material is easy to get, low in cost, therefore selects corn flour as carbon source.
The single factor test the selection result of nitrogen source shows that bacterium amount size is followed successively by yeast extract > CK under the conditions of different nitrogen sources
(combination of yeast extract and tryptone) > beef extract > peptone > soybean powder > tryptone > soya-bean cake > wheat bran > peanut cake >
Urea > ammonium sulfate；The size of bacteriostasis rate is followed successively by CK (combination of yeast extract and tryptone) > yeast extract > pancreas egg
White peptone > peptone > beef extract > urea > peanut cake > soya-bean cake > wheat bran > soybean powder > ammonium sulfate.Test discovery either biomass is also
It is bacteriostasis rate, the organic nitrogen source that nutrition is higher, nitrogen quantity is high will be significantly better than inorganic nitrogen-sourced.Control and yeast extract are produced in thallus
There was no significant difference in amount, but the bacteriostasis rate compareed is better than other nitrogen sources, therefore selects yeast extract and tryptone
Combination is used as nitrogen source.
The single factor test the selection result of inorganic salts shows that bacterium amount size is followed successively by MnSO under the conditions of different inorganic salts4>
K2HPO4>CaCO3>MgSO4>NaCl>CuSO4；The size of bacteriostasis rate is followed successively by K2HPO4>CaCO3>MgSO4>NaCl>MnSO4>
CuSO4.The MnSO in thallus yield4With K2HPO4Without significant difference, it is higher than other inorganic salts, is made due to testing with bacteriostatic activity
For main factor of evaluation, so selection K2HPO4As inorganic salts.
Orthogonal test: further to study the correlation between the nutrient source factor, corn flour has been determined in single factor experiment
2.0%, yeast extract 1.0%, tryptone 1.0%, K2HPO4On the basis of 0.5%, medium optimization L is carried out9(33) just
Experiment is handed over, as a result such as table 3.As can be seen from Table 3, the different factors combination of fermentation medium influences the fermentation of bacterial strain 140045 aobvious
It writes, when the combination of culture medium factor is A3B1C3D2, fermentation liquid bacteriostatic activity highest illustrates that the combination is conducive to bacterial strain 140045
Fermentation.It but is maximum value when A factor mean value 2, A factor level is 2.0% at this time；Reach maximum value when B factor mean value 2, at this time B
Factor level is 1.0%；Reach maximum value when C factor mean value 2, C factor level is 1.0% at this time；Reach when D factor mean value 2
Maximum value, D factor level is 0.5% at this time.It is most suitable for the culture medium factor that the fermentation of bacterial strain 140045 generates Substance
Group is combined into A2B2C2D2.It is respectively A > C > D > B according to the very poor size for finding out R of each component, corn flour is that bacterial strain 140045 ferments
Therefore biggest impact factor will control the dosage of corn flour during enzymatic production.
3 fermentation medium component orthogonal of table.
(2) optimum results of fermentation condition.
Single factor experiment.
Temperature fermentation conditional outcome shows that, when temperature is 24 DEG C~32 DEG C, biomass is improved as temperature increases；When
When temperature is 32 DEG C, biomass reaches maximum value；When temperature is higher than 32 DEG C, biomass is on a declining curve.For bacteriostasis rate, hair
Bacteriostasis rate is higher when existing temperature is between 24 DEG C~32 DEG C, and 28 DEG C reach maximum value, and when temperature is higher than 32 DEG C, bacteriostasis rate is significant
Decline.
Revolving speed fermentation condition the result shows that, when revolving speed be 100rpm~180rpm when, biomass and bacteriostasis rate are with revolving speed liter
It is high and increase；When revolving speed is 180rpmin, biomass and bacteriostatic activity reach maximum value；When revolving speed is higher than 200rpmin,
Biomass is on a declining curve with bacteriostasis rate.Therefore, the most suitable revolving speed that ferments is 180rpmin.
Inoculum concentration fermentation condition the result shows that, inoculum concentration be 1%~2% when, biomass and bacteriostasis rate increase with inoculum concentration
And it improves；When inoculum concentration is 2%, biomass and bacteriostasis rate reach maximum value；When inoculum concentration is higher than 2%, biomass and bacteriostasis rate
Start to increase and decline with inoculum concentration.Therefore, most suitable inoculum concentration of fermenting is 2%.
It is respectively 20mL, 35mL, 50mL, 65mL, 80mL in 150mL control bottling amount, fermentation results show when bottling amount
When for 20mL~80mL, biomass reduces, linear decline as bottling amount increases；When bottling amount is 20mL, biomass
Reach maximum value.For bacteriostasis rate, it is found that bacteriostasis rate is higher when bottling amount is 20mL~65mL, 50mL reaches maximum value, works as dress
Bacteriostasis rate is remarkably decreased when bottle amount is higher than 65mL.
Controlling pH is respectively 5,6,7,8,9, and when fermentation results show that pH is 5~7, biomass and bacteriostasis rate are with inoculum concentration
Increase and improves；When pH is 7, biomass and bacteriostasis rate reach maximum value；When pH is higher than 7, biomass and bacteriostasis rate start with pH
It increases and declines.Accordingly, it is determined that fermentation optimal pH is 7.
Control fermentation time be respectively for 24 hours, 48h, 72h, 96h, 120h, fermentation results show fermentation time be for 24 hours~
When 72h, biomass and bacteriostasis rate increase with fermentation time and are improved；When fermentation time is 72h, biomass and bacteriostasis rate reach most
Big value；When fermentation time is longer than 72h, biomass starts the extension with fermentation time with bacteriostasis rate and declines.Accordingly, it is determined that fermentation
Optimum time is 72.
Fermentation condition orthogonal test, for the correlation between further research fermentation condition, temperature, inoculation are chosen in test
Five amount, liquid amount, pH, fermentation time factors carry out medium optimization L25(55) orthogonal experiment, as a result such as table 4.It can be with by table 4
Find out, the different factor combination of fermentation condition influences significantly, when fermentation condition factor group is combined into the fermentation of bacterial strain 140045
A2B2C3D4E5, fermentation liquid bacteriostatic activity highest illustrates that the combination is conducive to the fermentation of bacterial strain 140045.
4 fermentation condition component orthogonal arrage of table.
Embodiment 3
The preparation of HMQAU140045 microbial bacterial agent.
(1) bacterial strain of China Committee for Culture Collection of Microorganisms's common micro-organisms center will actication of culture: be preserved in
HMQAU140045 (deposit number: CGMCC No.11768), in LB solid medium (tryptone 10g, yeast extract 5g,
Sodium chloride 5g, agar powder 15g, water 1000mL, pH7.0) 28 DEG C of culture 48h on plate, then it is transferred to LB solid slope culture medium
Upper 28 DEG C are cultivated for 24 hours, the strain activated.
(2) preparation of seed liquor prepares LB liquid medium (tryptone 10g, yeast extract 5g, chlorine according to a conventional method
Change sodium 5g, water 1000mL), 50mL culture medium, after 121 DEG C of moist heat sterilizations, every bottle of one collarium of access are packed into 150mL triangular flask
The strain of step (1) activation, 130rpm, 30 DEG C of oscillations for 24 hours, collect thallus, and be transferred to 108Cfu/mL1 prepares seed liquor.
(3) fermented and cultured fermentation medium (corn flour 20.0g, tryptone 10.0g, yeast extract 10.0g, phosphoric acid
Hydrogen dipotassium 0.5g is settled to 1000mL, pH 7.0 with distilled water), it is sub-packed in 500mL triangular flask, per bottled 200mL, 121 DEG C
It is spare to room temperature after cooling after moist heat sterilization, then the seed liquor of step (2) preparation is inoculated with and is inoculated in hair by inoculum concentration 1.5%
28 DEG C in ferment culture medium, 180r/min shake training 84h after, carry out microbial inoculum preparation.The preparation of A microbial inoculum, Optimal compositions of fermentation medium and fermentation
After condition shakes the fermentation liquid centrifugation that training obtains, thallus is washed twice with sterile water, and centrifugation, the thallus for scraping precipitating is placed in sterile water
In, it is configured to microbial inoculum A.The preparation of B microbial inoculum, the fermentation liquid obtained in optimal fermentation medium and fermentation condition for bacterial strain, 4 DEG C,
12000rpm is centrifuged 20 minutes acquisition supernatants, and by 0.22 μm of filter, collecting filtering liquid is microbial inoculum B.C: thallus+nothing
Fermented liquid preparation, after Optimal compositions of fermentation medium shakes the fermentation liquid centrifugation that training obtains, the thallus for scraping precipitating is placed in bacteria-free filtrate
In (not being diluted), it is configured to microbial inoculum C.
Embodiment 4
Inhibiting effect of the HMQAU140045 ferment filtrate to blueberry branch-rot bacterium.
(1) ferment filtrate tests blueberry branch-rot bacterium spore germination inhibiting effect
The fermentation medium and fermentation condition of antagonistic strain HMQAU 140045 are optimized, and in optimal conditions
The bacteria-free filtrate that fermentation obtains bacterial strain 140045 carries out following experiment.140045 pairs of vacations of bacterial strain HMQAU are observed using concave slide method
The influence of two born of the same parents' conidia germination of cocoa hair color, the two born of the same parents conidium angle of false cocoa hair color that picking blueberry limb is overflowed are used
Sterile water prepares spore suspension, concentration 106A/mL.140045 ferment filtrates are pressed to 50,100,150 and 200 times of dilution
Ratio is mixed with spore suspension respectively, respectively takes 40 μ L drops in the groove of concavity slide, the spore suspension configured with sterile water
Liquid is control.Slide is placed in 25 DEG C of constant-temperature moisture-keeping cultures in culture dish, observes spore germination situation after 4h under the microscope, is counted
Bacteriostasis rate is calculated, and carries out photomicrograph.Each processing sets 3 repetitions, which repeats 3 times.
(2) ferment filtrate tests the inhibiting effect that blueberry branch-rot bacterium mycelia grows
It is measured with 50,100,150,200 times of solid PDA medium mixed diluting to vacation using mycelial growth rate method
The inhibiting effect of two born of the same parents of cocoa hair color calculates bacteriostasis rate, the PDA culture medium of isometric aseptic culture fluid is added as compareing,
Each processing sets 5 repetitions.Simultaneously picking processing group and control group pathogen mycelia are tested, observes bacterial strain under an optical microscope
Influence of 140045 ferment filtrates to hypha form carries out photomicrograph.
(3) inhibiting effect result of the ferment filtrate to blueberry branch-rot bacterium spore germination
The ferment filtrate for diluting different multiples all has different degrees of inhibiting effect, fermentation filter to false two born of the same parents of cocoa hair color
Liquid dilutes 50 times of complete inhibition pathogen spore germinations, and inhibiting rate reaches 100%, and 100 times of dilution remains to reach 85% or more, with
Ferment filtrate extension rate increase inhibiting rate be gradually reduced.Microexamination discovery, the spore germination of control group is preferable, germ tube compared with
It is long, even thickness (Fig. 2-A)；Deformity cannot be sprouted or be sprouted to the spore of processing group, and germ tube top or centre are expanded, part spore
Content leaks (Fig. 2-B C D).
(4) the inhibiting effect result that ferment filtrate grows blueberry branch-rot bacterium mycelia
Bacterial strain HMQAU140045 ferment filtrate significantly inhibits the growth of false two born of the same parents mycelia of cocoa hair color, sends out
Ferment filtrate dilutes 50 times of complete inhibition pathogen mycelia growths, dilutes 100 times of inhibiting rates still close to 90%, as ferment filtrate is dilute
It releases multiple increase inhibiting rate to be gradually reduced, but dilutes 150 times and 200 times and the inhibiting rates of mycelia growth are remained to reach 71.25%
With 57.49% (table 5).Microexamination discovery, the mycelia thickness of control is consistent, branch is less, plasm is evenly distributed；Processing group
Interior other three groups of processing other than 50 times of dilution, although mycelia grows, mycelium, which expands thicker, lopsided, branch, to be increased, bacterium
Silk is gathered into warty, and plasm flows out to form ghost in some mycelia.
5 bacterial strain HMQAU140045 ferment filtrate of table grows the two born of the same parents' branch-rot bacterium spore germination of blueberry hair color and mycelia
Inhibit
Note: the same subsequent letter of column of figure indicates significant (DuncanShi duncan's new multiple range method) in 0.05 level difference of P <.
Embodiment 5
The measurement of antagonistic strain HMQAU140045 fermentation liquid antimicrobial spectrum.
Materials and methods: ferment filtrate is surveyed with 100 times of solid PDA medium mixed diluting using mycelial growth rate method
Determine to blueberry branch-rot bacterium, apple rot pathogen, Alternaria mali, apple anthrax bacteria, Apple Mould Core bacterium, Huang
15 kinds of melon target spot germ etc. the inhibiting effect for trying disease fungus calculate bacteriostasis rate, the PDA of isometric aseptic culture fluid is added
Culture medium sets 5 repetitions as control, each processing.
As a result with analysis: bacterial strain HMQAU140045 ferment filtrate antimicrobial spectrum measurement result show ferment filtrate dilution 100
There is different bacteriostatic activities to 15 kinds of test plant disease fungus after times.It is wherein best to the inhibitory effect of canker of apple fruit,
Inhibiting rate reaches 100%；Followed by Blueberry, grape seat chamber bacterium branch-rot bacterium, two born of the same parents' branch-rot bacterium of blueberry hair color, wheat root rotted leaf
Pinta bacterium, blueberry intend stem point branch-rot bacterium and fusarium graminearum, inhibiting rate are 76% or more；To other 7 kinds of pathogens
Inhibiting rate is also between 53.36%~70.73%；Inhibition to tomato wilt bacterium (trichothecium roseum) and Apple Mould Core bacterium
Rate is relatively poor, and respectively 40.22% and 35.45% or more.This illustrates that 140045 antimicrobial spectrum of bacterial strain is wider, antibacterial effect compared with
It is good, Biocontrol Potential with higher.
The inhibiting rate that 6 bacterial strain of table, 140045 fermentation liquid grows 15 kinds of pathogen mycelia
Note: the same subsequent letter of column of figure indicates significant (DuncanShi duncan's new multiple range method) in 0.05 level difference of P <.
Embodiment 6
The identification of bacterial strain HMQAU140045.
(1) form and physiological and biochemical property identification
Identification method is carried out referring to " the common bacteria system identification handbook " of the elegant pearl in east and Cai Miaoying.
(2) 16S rDNA molecular biology identification
Chelex100 method of the extraction of genomic DNA with reference to summer culvert etc..The PCR amplification and sequence of 16srDNA is analyzed: being mentioned
After taking strain gene group DNA, bacterial universal primers 27f (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5 '-is utilized
TACGGTTACCTTGTTACGACTT-3 ') PCR amplification is carried out to genomic DNA.Amplified production is through 1% agarose gel electrophoresis
After detection, purifying and bidirectional sequencing are carried out by Sangon Biotech (Shanghai) Co., Ltd., sequencing result is through Seqencher5.0
Contig (Contig) is exported after software automatic assembling and is carried out in NCBI (http://www.ncbi.nlm.gov) database
GenBank is submitted to after BLAST analysis.Suitable sequence is chosen from GenBank again, carries out sequence through 2.0 software of CLUSTALW
Column comparison, and systematic growth is constructed using adjacent method (neighbor-joininganalysis, NJ) with 5.05 software of MEGA
Tree, the number of repetition that wherein Bootstrap is examined are 1,000 time.
(3) form and physiological and biochemical property: bacterial strain HMQAU140045 30 DEG C of culture 2d, bacterium colony on LB solid medium
0.5~2.5mm of diameter, bacterium colony projection is creamy white, is opaque, is round, edge is irregular, there is fold on surface, in colony edge
There is crystalline material (Fig. 3-AB).Thallus is single or arrangement in pairs, Gram-positive, and rod-shaped (0.93 ± 0.24) μm~
(2.01 ± 0.52) μm, (Fig. 3-C) produce gemma.Physiology and biochemistry qualification result is shown in Table 7.It is identified by morphology and Physiology and biochemistry
The Preliminary Identification bacterium is bacillus amyloliquefaciens Bacillus amyloliquefaciens.
7 bacterial strain HMQAU140045 Physiology and biochemistry of table identifies table.
(4) Molecular Identification result: using bacterial universal primers 27f/1492r to the 16srDNA segment of bacterial strain 140045 into
Row PCR amplification determines segment overall length 1406bp through sequencing analysis.BLAST number in the sequence and GenBank that amplification is obtained
Tetraploid rice is carried out according to the nucleic acid sequence in library, the results showed that, bacterial strain HMQAU140045 and the solution starch from soil in Yunnan
The homology of bacillus amyloliquefaciens (GenBank accession number is JN99860) reaches 99%.System
Unite developmental analysis the result shows that, the sequence position of bacterial strain HMQAU140045 and 4 plants of Bacillus amyloliquefaciens bacterial strains
In the same branch (Fig. 4) of phylogenetic tree.Sequence analysis data and phylogenetic tree position further demonstrate bacterial strain
140045 be Bacillus amyloliquefaciens.
Embodiment 7
Inhibiting effect of the bacterial strain HMQAU140045 in vitro blueberry branch-rot.
Materials and methods: acquiring current year raw healthy blueberry branch (kind: Du Ke) from Qingdao Jiangnan blueberry planting base,
It is cut into the consistent branch section of thickness, after rinsing with ruinning water, with 75% ethyl alcohol surface sterilization, finally uses aseptic water washing.Test point four
Kind sprays medicament, and (A microbial inoculum is diluted to 1 × 108Cfu/mL, B microbial inoculum, C microbial inoculum are diluted to 1 × 108Cfu/mL, D: thiophanate-methyl
1500 times of dilutions), (1: application is inoculated with pathogen bacteria cake, 2 afterwards for 24 hours: to the slightly dry inoculation disease immediately of medicine after application for three kinds of processing
Opportunistic pathogen bacteria cake, 3: inoculation pathogen bacteria cake is administered afterwards for 24 hours), totally 12 kinds of combinations.
Two born of the same parents' bacteria cake of pathogen vacation cocoa hair color is inoculated with using needle point method, each branch is inoculated with 1 point, and each processing repeats
5 branches, to be inoculated with the branch of sterile water and liquid fermentation medium as control, test is repeated 3 times.It is inoculated with pathogen bacteria cake
Afterwards, it is fastened in bacteria cake with preservative film and moisturizing, condition of culture is 25 DEG C, 12h alternation of light and darkness.Guarantor is removed in culture afterwards for 24 hours
Fresh film simultaneously starts to observe and record incidence.Lesion size is measured after 7 days, calculate lesion area according to ellipse area formula and is prevented
Control effect；Control efficiency (%)=[(control lesion area-processing lesion area)/control lesion area] × 100%.
As a result with analysis: the bacteria suspension and ferment filtrate of bacterial strain HMQAU140045 has two born of the same parents' branch-rot of blueberry hair color
Apparent control efficiency, and better than control (table 8).The results show that application is inoculated with afterwards for 24 hours in 4 processing of pathogen, A (bacterium
Body) and C (fermentation liquid) preventive effect and D (thiophanate-methyl) preventive effect between without significant difference, inhibiting rate is 96% or more；B
The preventive effect of (ferment filtrate) remains to reach 93% or more less than first three processing.It is inoculated with pathogen simultaneously after application, is found
There was no significant difference between the preventive effect of B (ferment filtrate) and C (fermentation liquid) and the preventive effect of D (thiophanate-methyl), A (thallus) preventive effect
Significantly lower than other three kinds processing are 82.56%.Inoculation pathogen is administered afterwards for 24 hours, finds the preventive effect and D (methyl of C (fermentation liquid)
Thiophanate) preventive effect between there was no significant difference, D effect is best, followed by B (ferment filtrate), and A (thallus) effect is worst, suppression
Bacterium rate only has 67.06%.Therefore, application fermentation liquid (combination of thallus and fermentation liquid) effect, which is better than, is administered alone ferment filtrate
With thallus, be first administered be inoculated with afterwards pathogen effect better than first be inoculated with pathogen after be administered.
Control efficiency of 8 bacterial strain HMQAU140045 of table, the tri- kinds of microbial inoculums in vitro two born of the same parents' branch-rot of blueberry hair color
Note: the same subsequent letter of column of figure indicates significant (DuncanShi duncan's new multiple range method) in 0.05 level difference of P <.
Embodiment 8
Control efficiency of the bacillus amyloliquefaciens to two born of the same parents' branch-rot of blueberry hair color.
Materials and methods: in the blueberry base of Qingdao Jiangnan, choosing current year raw health and grow consistent blueberry branch,
It after 75% alcohol disinfecting, is stabbed with insect needle, wound diameter about 1-2mm, by false two born of the same parents of cocoa hair color in being activated in PDA culture medium
It after 24 hours, is beatened to take bacteria cake from colony edge with diameter 5mm punch, respectively thallus (the concentration of inoculating strain HMQAU140045
108Cfu/mL), fermentation liquid (concentration 108Cfu/mL) and ferment filtrate is micro- dry after by pure culture biscuits involvng inoculation thereon, moisturizing 24 hours,
To be inoculated with the branch of sterile water and thiophanate-methyl as control.Lesion size is measured after 7 days and calculates control efficiency.
As a result with analysis: test result shows that two kinds of microbial inoculums of ferment filtrate and fermentation liquid of bacterial strain HMQAU140045 are to indigo plant
The control efficiency of two born of the same parents' branch-rot of certain kind of berries hair color is preferable, and with the effect of thiophanate-methyl without significant difference (table 9).
Control efficiency of 9 bacterial strain HMQAU140045 of table, the tri- kinds of microbial inoculums to two born of the same parents' branch-rot of blueberry hair color
Note: the same subsequent letter of column of figure indicates significant (DuncanShi duncan's new multiple range method) in 0.05 level difference of P <.
Embodiment 9
The detection of bacterial strain HMQAU140045 induced enzyme.
Materials and methods:
For trying culture medium:
Chitinase detects culture medium: NH4H2PO41.0g, KCl 0.2g, MgSO4·7H2O 0.2g, tobacco brown spot pathogen 1%
(w/v) distilled water is settled to 1000mL, agar 20.0g, pH 7.0；
Protease detects culture medium: casein 10.0g, beef extract powder 3.0g, NaCl 5.0g, K2HPO42.0g, agar
15.0g, bromthymol blue 0.05g, pH 7.4 ± 0.1；
Cellulase detects culture medium: peptone 10.0g, yeast powder 10g, sodium carboxymethylcellulose 10.0g, NaCl
5.0g, KH2PO41.0g, distilled water are settled to 1000mL, agar 18.0g, pH 7.0；
β -1,3- dextranase detect culture medium: glucose 1.0g, Poria cocos powder 4.0g, K2HPO41.0g, Na2HPO4
3.0g, aniline blue 0.06g, FeSO4·7H2O 0.5g, agar powder 12g, distilled water are settled to 1000ml, pH 7.0；
Thermophilic iron element detects culture medium: chrome azurol S 60.5mg, 1mmol/L FeCl3·6H2O 10mL, cetyl front three
Base ammonium bromide 72.9mg, agar 20g, distilled water are settled to 1000mL, pH 7.0.
It detects whether 140045 thallus and ferment filtrate have enzyme activity, thallus detection: uses oese picking bacterial strain 140045
Thallus, point connect on five kinds of culture medium flat plates to be measured；Ferment filtrate detection: it is beaten around detection plate and takes three diameter 6mm's
The ferment filtrate that the HMQAU140045 of 20 μ L is obtained in Optimal compositions of fermentation medium and fermentation condition is added in circular hole in circular hole.28
DEG C culture 3d, observe chitinase, protease, β -1,3- dextranase detect culture medium flat plate whether there is or not transparent circles, observe thermophilic iron
Whether there is or not the generations of crocus haloing for element detection culture medium flat plate, generate corresponding enzyme if generating transparent circle or crocus haloing.It is fine
Tieing up plain enzyme detection observation method is the Congo red solution that appropriate 1mg/mL is added into the culture dish of culture 3d, dyes 1h, discards
Appropriate 1M NaCl solution is added in dye liquor, washs 1h, if generating cellulase, occurs around bacterium colony or ferment filtrate clear
Clear transparent circle.
As a result with analysis: induced enzyme testing result find, protease and cellulase detection culture medium flat plate on, bacterial strain
Transparent circle (Fig. 5) can be generated around HMQAU140045 and its ferment filtrate, shows that the bacterial strain can generate protease and fiber
Plain enzyme contains protease and cellulase in ferment filtrate, but in chitinase, β -1,3- dextranase, the detection training of thermophilic iron element
It supports and does not find corresponding transparent circle or crocus haloing on base plate, show that the bacterial strain cannot generate chitinase, β -1, the Portugal 3-
Dextranase and thermophilic iron element.
Embodiment 10
The heat stability test of bacterial strain HMQAU140045 ferment filtrate.
Bacterial strain HMQAU140045 ferment filtrate is handled into 60min in 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, 121 DEG C respectively, often
A processing is repeated 3 times.It is control with ferment filtrate without any processing after natural cooling.Each processing is in triplicate.With
False two born of the same parents of cocoa hair color are for trying bacterium, and using mycelial growth rate method measurement ferment filtrate, that treated at different conditions is antibacterial
Active (treated, and ferment filtrate dilutes 100 times in PDA culture medium) measures antibacterial work of each processing to pathogen respectively
Property.
As a result with analysis: as shown in Table 10, when temperature is within the scope of 40 DEG C~80 DEG C, Antagonistic Fungi ferment filtrate bacteriostasis rate with
Temperature raising is declined slightly, but bacteriostasis rate is still higher, is closer to compareing.At up to 100 DEG C, the antibacterial work of Antagonistic Fungi ferment filtrate
Property decline it is obvious, bacteriostasis rate reduces 27.40% relative to control；At 121 DEG C, the bacterium ferment filtrate bacteriostatic activity is almost complete
Portion loses, this shows that bioactive substance has stronger thermal stability in ferment filtrate, but high temperature can cause its activity sternly
Ghost image is rung, and also illustrates its active material such as protease and cellulase to having played important function in the process of inhibition of pathogen.
The thermal stability of 10 bacterial strain HMQAU140045 ferment filtrate of table
Note: the same subsequent letter of column of figure indicates significant (DuncanShi duncan's new multiple range method) in 0.05 level difference of P <
The embodiment above is the optimal embodiment of the present invention, but embodiment of the present invention is not by above-mentioned embodiment party
The limitation of case, it is other any without prejudice under conditions of the principle of the invention, can by way of changing parameter caused by reality
Example is applied, is all contained within protection scope of the present invention.
Nucleotide or amino acid sequence table
Claims (9)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain HMQAU140045, it is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are as follows: CGMCCNo.11768.
2. one kind is by bacillus amyloliquefaciens described in claim 1 (Bacillus amyloliquefaciens) bacterial strain
The microbial inoculum of HMQAU140045 preparation.
3. microbial inoculum according to claim 2, which is characterized in that the active constituent of microbial inoculum is bacillus amyloliquefaciens
The thallus and/or ferment filtrate of (Bacillus amyloliquefaciens) bacterial strain HMQAU140045.
4. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain described in claim 1
HMQAU140045 is for preventing and treating two born of the same parents' branch-rot of blueberry hair color.
5. microbial inoculum described in claim 2 or 3 is for preventing and treating two born of the same parents' branch-rot of blueberry hair color.
6. a kind of preparation method of microbial inoculum described in claim 2 or 3, which is characterized in that specific step is as follows:
(1) the HMQAU140045 bacterial strain of cryo-conservation is activated on LB solid medium, picking single colonie is in LB inclined-plane culture
It is cultivated 24 hours for 28 DEG C on base, obtains the strain of activation；
(2) strain for scraping ring activation from the inclined-plane LB with sterile oese, is inoculated in 50mL LB liquid medium
130rpm, 30 DEG C of oscillations for 24 hours, collect thallus, and adjusting cell concentration is 108Cfu/mL obtains seed liquor；
(3) seed liquor that step (2) obtains 1.5% is inoculated in fermentation medium by volume by fermented and cultured, in 28 DEG C,
180r/min shakes training 84h；
After shaking the fermentation liquid centrifugation that training obtains, thallus is washed twice with sterile water, and centrifugation, the thallus for scraping precipitating is placed in sterile water
In, preparation obtains the microbial inoculum A containing only thallus；
The obtained fermentation liquid of training is shaken in 4 DEG C, 12000rpm is centrifuged 20 minutes acquisition supernatants, by 0.22 μm of filter to get
To the microbial inoculum B containing only ferment filtrate；
After shaking the fermentation liquid centrifugation that training obtains, the thallus for scraping precipitating is placed in ferment filtrate, is obtained comprising thallus and fermentation filter
The microbial inoculum C of liquid.
7. the preparation method of microbial inoculum according to claim 6, which is characterized in that
The composition of the LB solid medium and LB slant medium are as follows: tryptone 10g, yeast extract 5g, sodium chloride
5g, agar powder 15g, water 1000mL, pH7.0；
The composition of the LB liquid medium are as follows: tryptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL,
pH7.0。
8. the preparation method of microbial inoculum according to claim 7, which is characterized in that the composition of the fermentation medium are as follows: beautiful
Rice flour 1.0-3.0%, yeast extract 0.5-1.5%, tryptone 0.5-1.5%, dipotassium hydrogen phosphate 0.3-0.7%,
PH7.0, surplus are water.
9. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain according to claim 1
HMQAU140045, which is characterized in that described for detecting bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
The method of bacterial strain HMQAU140045 is using the genomic DNA of strain to be tested as template, with 27f 5 '-
AGAGTTTGATCCTGGCTCAG-3 ' and 1492r 5 '-TACGGTTACCTTGTTACGACTT-3 ' is primer amplification 16SrDNA,
If gained amplified production is nucleotide sequence, as HMQAU140045 bacterial strain shown in SEQ ID No:1.
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