CN105733982A - Bacillus amyloliquefaciens used for preventing blueberry lasiodiplodia theobromae branch withering and inoculant and preparation method thereof - Google Patents
Bacillus amyloliquefaciens used for preventing blueberry lasiodiplodia theobromae branch withering and inoculant and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a strain of Bacillus amyloliquefaciens HMQAU140045 which is preserved in general microbiological center of China Committee for Culture Collection of Microorganisms in Institute of Microbiology, Chinese Academy of Sciences on December, 1st 2015 the preservation address is No.3, Yard 1, Beichen west road, Chaoyang district, Beijing, and the preservation number is CGMCC No.11768. An efficient microorganism is provided for blueberry lasiodiplodia theobromae branch withering, and a new way is opened up. The Bacillus amyloliquefaciens strain HMQAU140045 has a remarkable prevention effect on blueberry lasiodiplodia theobromae branch withering, and also has a good prevention effect on blueberry branch withering caused by other pathogenic bacteria.
Description
Technical field
The invention belongs to technical field of plant disease biological control, it is specifically related to the preventing and treating of a strain preventing and treating screening of bacillus amyloliquefaciens HMQAU140045 for blue berry hair color two born of the same parents' branch-rot, qualification, fermentation, Antibacterial Activity and blue berry hair color two born of the same parents' branch-rot, belongs to agricultural biological technical field.
Background technology
Blue berry belongs to Ericaceae Ericaceae, Vaccinium Vaccinium, is a kind of small berries fruit tree with high economic worth, because of the healthy nutritive value that it is prominent, is classified as one of big health food of the mankind five by FAO (Food and Agriculture Organization of the United Nation).Blue berry has become as a nationwide emerging leading industry, transform agricultural production for one, drive farmer richness, there is modern agriculture feature, and be the health industry of Chinese nation's health service, blue berry plantation industry also has become as one of the most popular industry of China's planting fruit trees industry.In the last few years, blue berry cultivated area also expands rapidly therewith, and national total area in 2015 reaches cultivation 31210 hectares, has increased sharply 51% than 15300 hectares in 2013.
But, along with the time limit expanded with plantation rapidly of cultivated area increases, the time limit increases, the reasons such as the miscarriage of technical experience shortage and individual casual household, the kind of blue berry disease increases increasingly and the extent of injury is on the rise, blue berry branch-rot has become as a kind of Major Diseases of restriction blue berry yield and quality at present, and it is also complicated various for causing the cause of disease kind of branch-rot, the Main Pathogenic Bacteria Fructus Vaccinii Bracteati Phomopsis Phomopsisvaccinii of current China Report, bar-shaped plan dish hirsutism Pestalotiopsisclavispora, Fructus Vitis viniferae seat chamber bacterium Botryosphaeriadothidea etc..In recent years, finding a kind of new branch-rot pathogenic fungi vacation cocoa hair color two born of the same parents Lasiodiplodiapseudotheobromae in the blue berry disease survey of Jiangnan, Qingdao of Shandong province, this disease infection rate is more rapid, blue berry has been produced and has constituted threat.And above branch-rot pathogen can individually be infected, also can Combined Infection.Discovery is investigated in preventing and treating for blue berry branch-rot, and field currently mainly or adopts Agro-chemicals control means, but food safety, pathogen Drug resistance and ecological disruption that life-time service chemical agent causes also will become the upper problems faced of production.Therefore finding a kind of new safely and effectively measure is the task of top priority to prevent and treat blue berry hair color two born of the same parents' branch-rot.
Biological control is as environmentally friendly, and preventive effect is stable, lasting, is not likely to produce drug-fast measure, will promote the sustainable development of agricultural.Nowadays, the research for Biological control has become as study hotspot both domestic and external, utilize biocontrol bacteria a kind of Main Means that controlling plant diseases is Biological control.Through retrieval, inventor does not retrieve bacillus amyloliquefaciens for the relevant report of blue berry hair color two born of the same parents' branch-rot Biological control and patent in existing patent and non-patent literature.
Summary of the invention
Present invention aim at providing the Bacillus amyloliquefaciens strain of preventing and treating blue berry hair color two born of the same parents' branch-rot, this bacterial strain has efficiently, the advantage that fungicidal spectrum is wide.
Realize technical scheme as described below.
One bacillus amyloliquefaciens BacillusamyloliquefaciensHMQAU140045, December in 2015 is preserved in Microbe Inst., Chinese Academy of Sciences on 1st China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number: CGMCCNo.11768.
Utilizing three kinds of microbial bacterial agents (A, B, C) that above-mentioned bacillus amyloliquefaciens HMQAU140045 produces, A active component is HMQAU140045 thalline, and B activity composition is ferment filtrate, and C active component is thalline and ferment filtrate.
One aspect of the present invention, comprises the microbial inoculum of bacillus amyloliquefaciens, and described microbial inoculum can be any microbial inoculum prepared by any technological means known to those skilled in the art.Such as by the concentrating agents of liquid form after fermentation culture, it is also possible to be the dry microbial inoculum obtained by lyophilizing.
A and C viable bacteria body number in mentioned microorganism microbial inoculum should be greater than 3.2x1010cfu/mL。
The preparation process of mentioned microorganism microbial inoculum is as follows:
(1) actication of culture, by the bacterial strain HMQAU140045 of low-temperature preservation on LB solid medium flat board 28 DEG C cultivate 48h, then be transferred on LB solid slant culture base 28 DEG C cultivate 24h, obtain activation strain；
(2) seed liquor prepares strain that aseptic inoculation one collarium step (1) activates in 50mLLB fluid medium 130rpm, 30 DEG C of vibration 24h, collects thalline, and is 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 DEG C by fermentation culture by volume, and 180r/min carries out microbial inoculum and prepares after shaking training 84h.Prepared by A microbial inoculum, shake the fermentation liquid that obtains of training centrifugal after, thalline sterilized water washes twice, centrifugal, and the thalline of scraping precipitation is placed in sterilized water, is configured to microbial inoculum A；Prepared by B microbial inoculum, fermentation liquid is in 4 DEG C, and 12000rpm obtains supernatant in centrifugal 20 minutes, by the filter of 0.22 μm, collects filtration liquid and is microbial inoculum B；Prepared by C microbial inoculum, after fermentation liquid is centrifugal, the thalline of scraping precipitation is placed in bacteria-free filtrate (not being diluted), is configured to microbial inoculum C.
The application in blue berry hair color two born of the same parents' branch-rot of the above-mentioned bacillus amyloliquefaciens.
The using method of microbial bacterial agent of the present invention, is that dilute with water cell concentration is to 1 × 10 by A in above-mentioned gained microbial bacterial agent8Cfu/mL, B are ferment filtrate, and C dilutes cell concentration to 1 × 10 with ferment filtrate8Cfu/mL, uses in blue berry branch-rot disease premorbid.
The screening and separating of HMQAU140045 bacterial strain: HMQAU140045 bacterial strain is that inventor separates from the blue berry rhizosphere soil of Qing Shi Huangdao District, Shandong Province and obtains.In April, 2014, inventor was from the blueberry garden of Huangdao District, Qingdao of Shandong province, and five point sampling methods gather Rhizosphere Soil, weigh 10g soil and add in 250 triangular flasks after mixing, add 90mL sterilized water, and vibrate 20min on shaking table, stands about 30 seconds, and supernatant is 10-1Diluent, then stepwise dilution becomes 10-2, 10-3, 10-4, 10-5Deng extension rate, after taking the diluent 1mL of rear three concentration and fusing, the LB culture medium 9mL of cooling is mixed into flat board, each concentration is in triplicate, cultivate 1-3d at 28 DEG C, carry out separation and the purification of antibacterial, and with blue berry hair color two born of the same parents branch-rot bacterium for target, stood facing each other method by flat board, spore germination method and isolated shoot test carry out the screening of antagonistic strain, and blue berry hair color two born of the same parents' branch-rot is had the bacterial strain of obvious prevention effect, called after HMQAU140045 by final acquisition one strain.
The taxonomic identification of HMQAU140045 microbial inoculum:
(1) identification by morphological characters
Cultivating 2d, colony diameter 0.5～2.5mm for 30 DEG C on LB solid medium, bacterium colony projection is creamy white, opaque, circular, edge is irregular, there is fold on surface, has crystalline material at colony edge.Thalline Gram-positive, shaft-like, (0.93 ± 0.24) μm～(2.01 ± 0.52) μm, single or arrange in pairs, produce spore.Physiological and biochemical test result shows that this bacterial strain is facultative anaerobe, starch, reduction nitrate can be hydrolyzed, can utilizing citrate, glucose, fructose, sucrose, mannitol, catalase, oxidase and V-P reaction are the positive, and denitrification and clark and Lubsreaction are negative.This bacterial strain has toleration under the acid condition of 2%～7%NaCl and pH5, does not have toleration under the alkali condition of 10%NaCl and pH9；4 DEG C can not grow, and 51 DEG C can grow.Identify that this bacterium of Preliminary Identification is bacillus amyloliquefaciens Bacillusamyloliquefaciens by morphology and Physiology and biochemistry；
(2) Molecular Identification
With HMQAU140045 genomic DNA for template, utilize primer 2 7f/1492r that 16srDNA fragment is expanded.Described primer sequence is 27f (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5 '-TACGGTTACCTTGTTACGACTT-3 ').Amplification reaction system: each 1 μ L of 10 × Buffer2.5 μ L, 5mMdNTP2 μ L, 10 μMs of primer 2 7f and primer 1492r, Taq enzyme (5U/ μ L) 0.5 μ L, template DNA 2.5 μ L, moisturizing is to 25 μ L.Reaction condition: 94 DEG C of denaturation 5min；94 DEG C of degeneration 1min, 53 DEG C of annealing 1min, 72 DEG C extend 1.5min, 30 circulations；Last 72 DEG C extend 10min.Amplified production is after 1% agarose gel electrophoresis detection, being purified and two-way order-checking by Sangon Biotech (Shanghai) Co., Ltd., sequencing result submits to GenBank after deriving contig (Contig) after Sequencher5.0 software assembles automatically and carrying out BLAST analysis in NCBI (http://www.ncbi.nlm.gov) data base.Suitable sequence is chosen again from GenBank, carry out alignment through CLUSTALW2.0 software, and adopt adjacent method (neighbor joininganalysis, NJ) phylogenetic tree construction with MEGA5.05 software, wherein the number of repetition of Bootstrap inspection is 1,000 time.It is shown that this bacterial strain and the homology from the bacillus amyloliquefaciens Bacillusamyloliquefaciens (GenBank accession number is JN99860) of soil in Yunnan reach 99%.Phylogenetic Analysis is it is shown that the sequence of this bacterial strain and 4 strain Bacillusamyloliquefaciens bacterial strains is positioned at the same branch of phylogenetic tree.It is Bacillusamyloliquefaciens that sequence analysis data and phylogenetic tree position further demonstrate bacterial strain HMQAU140045.
Comprehensive above morphological characteristic and 16SrDNA sequence homology comparison are analyzed the known HMQAU140045 of result and are belonged to bacillus amyloliquefaciens (Bacillusamyloliquefaciens), and different with existing Bacillus amyloliquefaciens strain, it is a new strains.
The authentication method of above-mentioned bacillus amyloliquefaciens strain HMQAU140045, including with the genomic DNA of test strains for template, with 27f and the 1492r amplification being primer, if gained PCR primer is the nucleotide sequence shown in SEQIDNo:1, it is HMQAU140045 bacterial strain.
The B microbial inoculum of above-mentioned Bacillus amyloliquefaciens strain HMQAU140045 and the detection of C microbial inoculum, detecting culture medium detection with protease and cellulase, if there being positive reaction, being B microbial inoculum and the C microbial inoculum of HMQAU140045 bacterial strain.
Advantages of the present invention and beneficial effect: (1) present invention is that blue berry hair color two born of the same parents' branch-rot provides an efficient microorganism, opens a new approach；(2) blue berry hair color two born of the same parents' branch-rot is had notable preventive effect by Bacillus amyloliquefaciens strain HMQAU140045 of the present invention, and other cause of disease microbial blue berry branch-rot also has the enforcement of good preventive effect.(3) microbial bacterial agent of the present invention is to people, animal safety；It is not likely to produce Drug resistance, reduces resistance risk；For environmentally friendly, using of chemical bactericide can be reduced, reduce the destruction on environment and the impact on human health, to realize the sustainable development safely and efficiently that blue berry produces.(4) low production cost of the present invention, preparation method and using method are simple.
Accompanying drawing explanation
The present invention has drawings described below:
Fig. 1 is the Bacillus amyloliquefaciens strain HMQAU140045 antagonistic effect to blue berry hair color two born of the same parents branch-rot bacterium；
Fig. 2 is the impact on false cocoa hair color two born of the same parents' conidia germination of the Bacillus amyloliquefaciens strain HMQAU140045 ferment filtrate；
Fig. 3 is Bacillus amyloliquefaciens strain HMQAU140045 colonial morphology and Gram’s staining；
Fig. 4 is the antagonistic strain Bacillus amyloliquefaciens strain HMQAU140045 based on 16SrDNA gene order structure and NJ (neighbor-joininganalysis) phylogenetic tree of Related Bacteria；
Fig. 5 is protease and the cellulase testing result of Bacillus amyloliquefaciens strain HMQAU140045.
Detailed description of the invention
Case study on implementation is used for being explained further the present invention below, but is construed as limiting the invention never in any form.Test method in following case study on implementation, if no special instructions, is generally conventional method；Percentage composition in following case study on implementation, if no special instructions, is weight percentage.
Embodiment 1
The separation of antagonistic strain and screening.
(1) separate: in April, 2014 gathers the Rhizosphere Soil of blue berry plant in the blueberry garden of Huangdao District, Qingdao of Shandong province.Adopting dilution-plate method isolated bacterial strain, culture medium is LB solid medium, and after growing bacterium colony, picking antibacterial list bacterium colony is in LB solid plate line purification and is stored in mycology research department of Qingdao Agricultural University.
(2) primary dcreening operation: with false cocoa hair color two born of the same parents for target, adopts face-off flat band method, PDA plate central authorities streak inoculation antibacterial, and the two ends inoculation pathogen bacterium cake from antibacterial 2.5cm, 25 DEG C of constant temperature culture, comparison is inoculation pathogen bacterium cake only.Each process sets 3 repetitions, when 3/4 culture dish is covered with in comparison, measures antibacterial bandwidth, filters out the bacterial isolates that false cocoa hair color two born of the same parents have obvious antagonism.
(3) multiple sieve: the bacterial isolates after primary dcreening operation prepares seed liquor, takes 1mL seed liquor and is inoculated in 50mLLB culture medium, 130r/min, 30 DEG C of constant-temperature shaking culture 48h.Being centrifuged by fermentation liquid, the bacteria-free filtrate of Antagonistic Fungi prepared by 0.22 μm of filtering with microporous membrane.Adopt mycelial growth rate method, by PDA culture medium (45～50 DEG C) mixing of cooling after fermentation liquid bacteria-free filtrate and thawing, it is down flat plate, to add the PDA culture medium of equal-volume LB fluid medium as comparison, inoculate, in flat board central authorities, false cocoa hair color two born of the same parents' pathogenic bacteria bacterium cake that diameter is 5mm.Each process sets 3 repetitions, and when 3/4 culture dish is covered with in comparison, decussation method measures matched group and process group colony diameter, calculates bacteriostasis rate, filters out the bacterial isolates that false cocoa hair color two born of the same parents' antagonistic effect is optimum.
Suppression ratio (%)=(comparison bacterium colony half (mm)-process colony radius (mm))/comparison colony radius (mm) × 100%
Test adopt simultaneously both the above method measure the best antagonistic strain of the fungistatic effect that obtains of screening to other bar-shaped plan dish hirsutism Pestalotiopsisclavispora of three strains blue berry branch-rot bacterium, Fructus Vitis viniferae seat chamber bacterium Botryosphaeriadothidea, Fructus Vaccinii Bracteati Phomopsis Phomopsisvaccinii inhibitory action.
(4) result and analysis: utilize dilution-plate method to separate from the Rhizosphere Soil of healthy blue berry plant and obtain 20 strain antibacterials, face-off flat band method filters out 7 strains the antagonistic strain of obvious inhibition to false cocoa hair color two born of the same parents, account for the 35% of institute's isolated strains, wherein bacterial strain HMQAU140045 antagonistic effect is best, and antibacterial bandwidth reaches 14.50mm (Fig. 1-A).Choosing the LB ferment filtrate of this bacterial strain to dilute 10 times in PDA culture medium and carry out mycelial growth rate method test, result shows that the suppression ratio of false cocoa hair color two born of the same parents' mycelial growth can be reached 100% (Fig. 1-B) by the ferment filtrate of this bacterial strain.Additionally, antibacterial bandwidth respectively 12.63mm, 18.3mm, 8.75mm that bacterial strain HMQAU140045 is to Fructus Vitis viniferae seat chamber bacterium, Fructus Vaccinii Bracteati Phomopsis and bar-shaped plan dish stey.This bacterial strain dilutes the LB ferment filtrate of 10 times to the suppression ratio of Fructus Vitis viniferae seat chamber bacterium, Fructus Vaccinii Bracteati Phomopsis and bar-shaped plan dish stey respectively 100%, and 89.67%, 76.74%.Illustrate that multiple blue berry branch-rot pathogen is had good fungistatic effect by bacterial strain HMQAU140045.
Embodiment 2
The optimization of antagonistic strain HMQAU140045 fermentation medium and condition.
(1) optimization of fermentation medium
Adopt the optimization method of single factor test and orthogonal test, first pass through single factor experiment (carbon source, nitrogenous source, inorganic salt) and determine the nutrient media components of the best, the appropriate value level of each component of reselection, carry out orthogonal test.Test with false cocoa hair color two born of the same parents for for examination pathogen.Accessing after seed liquor 30 DEG C, 130r/min shakes training 48h., with the impact on HMQAU140045 thalline generation amount of the spectrophotometric determination difference factor.Mycelial growth rate method is adopted to measure the ferment filtrate bacteriostasis rate (carbon and nitrogen sources ferment filtrate dilutes 50 times, other dilution 100 times) to false cocoa hair color two born of the same parents.
Single factor test optimizes.
Carbon source optimizing: with glucose, sucrose, maltose, fructose, D-xylose, lactose, soluble starch, potato starch, Semen Maydis powder for for examination carbon source, basal medium (LB culture medium) adds the carbon source of 2%, each carbon source sets three repetitions, with the basal medium that is not added with carbon source for comparison.
Nitrogenous source optimizes: with ammonium sulfate, carbamide, soybean cake, peanut cake, analysis for soybean powder, Carnis Bovis seu Bubali cream, peptone, tryptone, yeast powder.Adding optimum carbon source in basal medium (LB culture medium) as comparison (CK), respectively with the tryptone in 2.0% nitrogenous source equivalent substitution control medium and yeast extract, each process repeats for 3 times.
Inorganic salt optimizes: with CuSO4、MnSO4、MgSO4、CaCO3、K2HPO4For for examination inorganic salt, adding NaCl for comparison with the suitableeest carbon nitrogen source, replace the NaCl in control medium with 0.5% inorganic salt equivalent respectively.
Orthogonal optimization: the optimum carbon source that filters out with single factor experiment, nitrogenous source, inorganic salt, for variance factor, select the 4 horizontal L of factor 39(34) orthogonal table carries out medium optimization test, it is determined that the optimum proportioning (table 1) of each component of culture medium.
(2) optimization of fermentation condition
The optimization of fermentation condition is divided into single factor experiment (rotating speed, temperature, initial pH, incubation time, inoculum concentration and liquid amount) and orthogonal test, on the basis of single factor experiment, suitable level is selected to carry out orthogonal test, it is determined that optimal conditions of fermentation combines.Test with false cocoa hair color two born of the same parents for for examination pathogen.
Table 1 fermentation medium compositional factors water-glass.
Single factor experiment: when optimization obtains Optimal compositions of fermentation medium, determining pH (5.0,6.0,7.0,8.0 and 9.0), cultivation temperature (24,28,32,36 and 40 DEG C), rotating speed (100,120,140,160,180 and 200rpm), liquid amount (20,30,40,50 and 60mL), the initial inoculum (1,2,4, the 6 and 8%) impact on bacteriostatic activity, each process repeats for three times.Assay method is with fermentation medium optimization (ferment filtrate dilutes 100 times).
Orthogonal test: adopt Optimal Medium, when the suitableeest rotating speed, with cultivation temperature, initial pH, incubation time, inoculum concentration and liquid amount for investigating factor, by orthogonal array L25(55) 5 factors of design, 5 levels orthogonal test (table 2), the relatively various fermentation conditions combination impact on the bacteria-free filtrate antibacterial activity of biocontrol bacterial strain fermentation liquid, it is determined that the most adaptable method.Assay method is with fermentation medium optimization (ferment filtrate dilutes 100 times).
Table 2 fermentation condition factor level table.
(1) optimum results of fermentation medium.
Single factor test optimizes.
The single factor test the selection result of carbon source shows, when Semen Maydis powder is as carbon source, after fermentation, bacterium amount is maximum, and bacterium amount size is followed successively by maltose thereafter > potato starch > lactose > soluble starch > D-xylose > glucose > sucrose > fructose > it is not added with carbon source；The size of bacteriostasis rate is followed successively by maltose > Semen Maydis powder > potato starch > soluble starch > lactose > D-xylose > sucrose > glucose > fructose > it is not added with carbon source.When finding using Semen Maydis powder as carbon source, although bacteriostasis rate is not as good as maltose, but there was no significant difference simultaneously, and Semen Maydis powder as carbon source time bacterium amount the highest, and material is easy to get, with low cost, therefore selects Semen Maydis powder as carbon source.
The single factor test the selection result of nitrogenous source shows, when different nitrogen sources, bacterium amount size is followed successively by yeast extract > CK (combination of yeast extract and tryptone) > Carnis Bovis seu Bubali cream > peptone > analysis for soybean powder > tryptone > soybean cake > Testa Tritici > peanut cake > carbamide > ammonium sulfate；The size of bacteriostasis rate is followed successively by CK (combination of yeast extract and tryptone) > yeast extract > tryptone > peptone > Carnis Bovis seu Bubali cream > carbamide > peanut cake > soybean cake > Testa Tritici > analysis for soybean powder > ammonium sulfate.No matter test is biomass or bacteriostasis rate if finding, the organic nitrogen source that nutrition is higher, nitrogen quantity is high will significantly better than inorganic nitrogen-sourced.Comparison and yeast extract there was no significant difference in thalline yield, but the bacteriostasis rate of comparison is better than other nitrogenous sources, therefore select the yeast extract combination with tryptone as nitrogenous source.
The single factor test the selection result of inorganic salt shows, when different inorganic salt, bacterium amount size is followed successively by MnSO4>K2HPO4>CaCO3>MgSO4>NaCl>CuSO4；The size of bacteriostasis rate is followed successively by K2HPO4>CaCO3>MgSO4>NaCl>MnSO4>CuSO4.MnSO in thalline yield4With K2HPO4Without significant difference, higher than other inorganic salts, owing to test is using bacteriostatic activity as main factor of evaluation, so selecting K2HPO4As inorganic salt.
Orthogonal test: for studying the dependency between the nutrient source factor further, determines Semen Maydis powder 2.0%, yeast extract 1.0%, tryptone 1.0%, K at single factor experiment2HPO4On the basis of 0.5%, carry out medium optimization L9(33) orthogonal experiment, result is table 3 such as.As can be seen from Table 3, the different factors combine of fermentation medium are notable on bacterial strain 140045 fermentation impact, when culture medium factor is combined as A3B1C3D2, fermentation liquid bacteriostatic activity is the highest, and this fermentation being combined with being beneficial to bacterial strain 140045 is described.But being maximum during A factor average 2, now A factor level is 2.0%；Reaching maximum during B factor average 2, now B factor level is 1.0%；Reaching maximum during C factor average 2, now C factor level is 1.0%；Reaching maximum during D factor average 2, now D factor level is 0.5%.The culture medium factor being best suitable for bacterial strain 140045 fermentation generation Substance is combined as A2B2C2D2.Find out the size respectively A > C > D > B of R according to each component extreme difference, Semen Maydis powder is maximum effect factor of bacterial strain 140045 fermentation, therefore, to control the consumption of Semen Maydis powder in enzymatic production process well.
Table 3 fermentation medium component orthogonal.
(2) optimum results of fermentation condition.
Single factor experiment.
Temperature fermentation conditional outcome shows, when temperature is 24 DEG C～32 DEG C, biomass raises along with temperature and improves；When temperature is 32 DEG C, biomass reaches maximum；When temperature is higher than 32 DEG C, biomass is on a declining curve.For bacteriostasis rate, it has been found that when temperature is between 24 DEG C～32 DEG C, bacteriostasis rate is higher, 28 DEG C reach maximum, and when temperature is higher than 32 DEG C, bacteriostasis rate is remarkably decreased.
Rotating speed fermentation condition is it is shown that when rotating speed is 100rpm～180rpm, biomass raises with rotating speed with bacteriostasis rate and raises；When rotating speed is 180rpmin, biomass and bacteriostatic activity reach maximum；When rotating speed is higher than 200rpmin, biomass is on a declining curve with bacteriostasis rate.Therefore, the suitableeest rotating speed that ferments is 180rpmin.
Inoculum concentration fermentation condition is it is shown that when inoculum concentration is 1%～2%, biomass increases with inoculum concentration with bacteriostasis rate and improves；When inoculum concentration is 2%, biomass and bacteriostasis rate reach maximum；When inoculum concentration is higher than 2%, biomass and bacteriostasis rate start to raise with inoculum concentration and decline.Therefore, the suitableeest inoculum concentration of fermenting is 2%.
Controlling bottling at 150mL and measure respectively 20mL, 35mL, 50mL, 65mL, 80mL, fermentation results shows, when bottling amount is 20mL～80mL, biomass raises along with bottling amount and reduces, linear decline；When bottling amount is 20mL, biomass reaches maximum.For bacteriostasis rate, it has been found that when bottling amount is 20mL～65mL, bacteriostasis rate is higher, and 50mL reaches maximum, when bottling amount is higher than 65mL, bacteriostasis rate is remarkably decreased.
Controlling pH respectively 5,6,7,8,9, fermentation results shows, when pH is 5～7, biomass increases with inoculum concentration with bacteriostasis rate and improves；When pH is 7, biomass and bacteriostasis rate reach maximum；When pH is higher than 7, biomass and bacteriostasis rate start to raise with pH and decline.Accordingly, it is determined that fermentation optimum pH is 7.
Controlling fermentation time respectively 24h, 48h, 72h, 96h, 120h, fermentation results shows, when fermentation time is 24h～72h, biomass increases with fermentation time with bacteriostasis rate and improves；When fermentation time is 72h, biomass and bacteriostasis rate reach maximum；When fermentation time is longer than 72h, biomass and bacteriostasis rate start to decline with the prolongation of fermentation time.Accordingly, it is determined that fermentation optimum time is 72.
Fermentation condition orthogonal test, for studying the dependency between fermentation condition further, temperature is chosen in test, inoculum concentration, liquid amount, pH, five factors of fermentation time carry out medium optimization L25(55) orthogonal experiment, result is table 4 such as.As can be seen from Table 4, the different factors combine of fermentation condition is notable on bacterial strain 140045 fermentation impact, when fermentation condition factors combine is A2B2C3D4E5, fermentation liquid bacteriostatic activity is the highest, and this fermentation being combined with being beneficial to bacterial strain 140045 is described.
Table 4 fermentation condition component orthogonal table.
Embodiment 3
The preparation of HMQAU140045 microbial bacterial agent.
(1) actication of culture: the bacterial strain HMQAU140045 (deposit number: CGMCCNo.11768) at China Committee for Culture Collection of Microorganisms's common micro-organisms center will be preserved in, in LB solid medium (tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL, pH7.0) on flat board 28 DEG C cultivate 48h, be transferred on LB solid slant culture base again 28 DEG C cultivate 24h, obtain activation strain.
(2) LB fluid medium (tryptone 10g is prepared in the preparation of seed liquor according to a conventional method, yeast extract 5g, sodium chloride 5g, water 1000mL), 150mL triangular flask loads 50mL culture medium, after 121 DEG C of moist heat sterilizations, every bottle graft enters the strain that a collarium step (1) activates, 130rpm, 30 DEG C of vibration 24h, collect thalline, and be transferred to 108Cfu/mL1, prepares seed liquor.
(3) fermentation culture fermentation medium (Semen Maydis powder 20.0g, tryptone 10.0g, yeast extract 10.0g, dipotassium hydrogen phosphate 0.5g, it is settled to 1000mL, pH7.0 with distilled water), it is sub-packed in 500mL triangular flask, every bottled 200mL,, after 121 DEG C of moist heat sterilizations, standby to room temperature after cooling, again prepared by step (2) seed liquor inoculation by inoculum concentration 1.5% be inoculated in fermentation medium 28 DEG C, 180r/min shake training 84h after, carry out microbial inoculum and prepare.Prepared by A microbial inoculum, Optimal compositions of fermentation medium and fermentation condition shake the fermentation liquid that obtains of training centrifugal after, thalline sterilized water washes twice, centrifugal, and the thalline of scraping precipitation is placed in sterilized water, is configured to microbial inoculum A.Prepared by B microbial inoculum, for the fermentation liquid that bacterial strain obtains at optimum fermentation medium and fermentation condition, at 4 DEG C, centrifugal 20 minutes acquisition supernatant of 12000rpm, by the filter of 0.22 μm, collection is filtered liquid and is microbial inoculum B.C: thalline+prepare without fermented liquid, Optimal compositions of fermentation medium shake the fermentation liquid that obtains of training centrifugal after, the thalline of scraping precipitation is placed in bacteria-free filtrate (not being diluted), is configured to microbial inoculum C.
Embodiment 4
The HMQAU140045 ferment filtrate inhibitory action to blue berry branch-rot bacterium.
(1) blue berry branch-rot bacterium spore germination inhibitory action is tested by ferment filtrate
Fermentation medium and fermentation condition to antagonistic strain HMQAU140045 are optimized, and the bacteria-free filtrate obtaining bacterial strain 140045 that ferments in optimal conditions carries out following experiment.Adopting concave slide method to observe the bacterial strain HMQAU140045 impact on false cocoa hair color two born of the same parents' conidia germination, the false cocoa hair color two born of the same parents conidium angle that picking blue berry branch is overflowed, prepare spore suspension with sterilized water, concentration is 106Individual/mL.140045 ferment filtrates are mixed with spore suspension respectively by the dilution ratio of 50,100,150 and 200 times, respectively takes in the 40 μ L groove dropping in concavity slide, with the spore suspension of sterilized water configuration for comparison.Being placed in culture dish by slide 25 DEG C of constant-temperature moisture-keepings cultivates, and examines under a microscope spore germination situation, calculate bacteriostasis rate, and carry out photomicrograph after 4h.Each process sets 3 repetitions, and this test repeats 3 times.
(2) inhibitory action of blue berry branch-rot bacterium mycelial growth is tested by ferment filtrate
Adopt mycelial growth rate method that itself and solid PDA medium mixed diluting 50,100,150,200 times are measured the inhibitory action to false cocoa hair color two born of the same parents, calculate bacteriostasis rate, to add the PDA culture medium of equal-volume aseptic culture fluid as comparison, each process sets 5 repetitions.Experiment picking process group and matched group pathogen mycelia, observe the impact on hypha form of bacterial strain 140045 ferment filtrate under an optical microscope, carry out photomicrograph.
(3) ferment filtrate inhibitory action result to blue berry branch-rot bacterium spore germination
False cocoa hair color two born of the same parents are had inhibitory action in various degree by the ferment filtrate of dilution different multiples, ferment filtrate dilutes 50 times and suppresses pathogen spore germination completely, suppression ratio reaches 100%, dilute 100 times to remain to reach more than 85%, be gradually reduced along with ferment filtrate extension rate increases suppression ratio.Microexamination finds, the spore germination of matched group is better, and germ tube is longer, even thickness (Fig. 2-A)；The spore of process group can not sprout or sprout deformity, and germ tube top or centre are expanded, and part spore content leaks (Fig. 2-BCD).
(4) ferment filtrate inhibitory action result to blue berry branch-rot bacterium mycelial growth
False cocoa hair color two born of the same parents' mycelial growth is had obvious inhibitory action by bacterial strain HMQAU140045 ferment filtrate, ferment filtrate dilutes 50 times and suppresses pathogen mycelial growth completely, dilute 100 times of suppression ratio still close to 90%, it is gradually reduced along with ferment filtrate extension rate increases suppression ratio, but dilution 150 times and 200 times of suppression ratio to mycelial growth remain to reach 71.25% and 57.49% (table 5).Microexamination finds, the mycelia thickness of comparison is consistent, branch is less, protoplasm is evenly distributed；Other three groups process outer except dilution 50 times in process group, although mycelial growth, but mycelium expands thicker, lopsided, branch increases, and mycelia is gathered into warty, protoplasm outflow formation ghost in some mycelia.
The suppression to blue berry hair color two born of the same parents' branch-rot bacterium spore germination and mycelial growth of the table 5 bacterial strain HMQAU140045 ferment filtrate
Note: same column of figure letter representation below is at P < 0.05 level difference notable (DuncanShi duncan's new multiple range method).
Embodiment 5
The mensuration of antagonistic strain HMQAU140045 fermentation liquid antimicrobial spectrum.
Materials and methods: adopt mycelial growth rate method to be measured blue berry branch-rot bacterium, apple rot pathogen, Alternaria mali, apple anthrax bacteria, Apple Mould Core bacterium, Leaf Spot Caused by Corynespora cassiicola on Cucumber bacterium etc. 15 kinds for the inhibitory action trying pathogenic fungi with solid PDA medium mixed diluting 100 times by ferment filtrate, calculate bacteriostasis rate, to add the PDA culture medium of equal-volume aseptic culture fluid as comparison, each process sets 5 repetitions.
Result and analysis: bacterial strain HMQAU140045 ferment filtrate antimicrobial spectrum measurement result shows, 15 kinds of test plant pathogenic fungi are had different bacteriostatic activities after diluting 100 times by ferment filtrate.Wherein that the inhibition of canker of apple fruit is best, it is suppressed that rate reaches 100%；Next to that Blueberry, grape seat chamber bacterium branch-rot bacterium, blue berry hair color two born of the same parents branch-rot bacterium, Wheat Leaf Spot Pathogen Bipolaris Sorokiniana, blue berry plan stem point branch-rot bacterium and fusarium graminearum, it is suppressed that rate is more than 76%；To the suppression ratio of other 7 kinds of pathogen also between 53.36%～70.73%；The suppression ratio of tomato wilt bacterium (trichothecium roseum) and Apple Mould Core bacterium is relatively poor, respectively 40.22% and more than 35.45%.This illustrates that bacterial strain 140045 antimicrobial spectrum is relatively wide, and antibacterial effect is better, has higher Biocontrol Potential.
Table 6 bacterial strain 140045 fermentation liquid suppression ratio to 15 kinds of pathogen mycelial growths
Note: same column of figure letter representation below is at P < 0.05 level difference notable (DuncanShi duncan's new multiple range method).
Embodiment 6
The qualification of bacterial strain HMQAU140045.
(1) form and physiological and biochemical property are identified
Authentication method carries out with reference to " the common bacteria system identification handbook " of the elegant pearl in east and Cai Miaoying.
(2) 16SrDNA molecular biology identification
The chelex100 method extracting reference Xia Han etc. of genomic DNA.The pcr amplification of 16srDNA and sequence analysis: extract after strain gene group DNA, utilize bacterial universal primers 27f (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5 '-TACGGTTACCTTGTTACGACTT-3 ') that genomic DNA is carried out pcr amplification.Amplified production is after 1% agarose gel electrophoresis detection, being purified and two-way order-checking by Sangon Biotech (Shanghai) Co., Ltd., sequencing result submits to GenBank after deriving contig (Contig) after Seqencher5.0 software assembles automatically and carrying out BLAST analysis in NCBI (http://www.ncbi.nlm.gov) data base.Suitable sequence is chosen again from GenBank, carry out alignment through CLUSTALW2.0 software, and adopt adjacent method (neighbor-joininganalysis, NJ) phylogenetic tree construction with MEGA5.05 software, wherein the number of repetition of Bootstrap inspection is 1,000 time.
(3) form and physiological and biochemical property: bacterial strain HMQAU140045 is 30 DEG C of cultivation 2d on LB solid medium, colony diameter 0.5～2.5mm, bacterium colony projection is creamy white, opaque, circular, edge is irregular, there is fold on surface, has crystalline material (Fig. 3-AB) at colony edge.Thalline is single or arranges in pairs, Gram-positive, shaft-like (0.93 ± 0.24) μm～(2.01 ± 0.52) μm, and (Fig. 3-C) produces spore.Physiology and biochemistry qualification result is in Table 7.Identify that this bacterium of Preliminary Identification is bacillus amyloliquefaciens Bacillusamyloliquefaciens by morphology and Physiology and biochemistry.
Table 7 bacterial strain HMQAU140045 Physiology and biochemistry identifies table.
(4) Molecular Identification result: utilize bacterial universal primers 27f/1492r that the 16srDNA fragment of bacterial strain 140045 is carried out pcr amplification, through sequencing analysis, it is determined that this fragment total length 1406bp.Sequence amplification obtained carries out tetraploid rice with the nucleotide sequence in BLAST data base in GenBank, it is shown that the homology of bacterial strain HMQAU140045 and the bacillus amyloliquefaciens Bacillusamyloliquefaciens (GenBank accession number is JN99860) from soil in Yunnan reaches 99%.Phylogenetic Analysis is it is shown that the sequence of bacterial strain HMQAU140045 and 4 strain Bacillusamyloliquefaciens bacterial strains is positioned at the same branch (Fig. 4) of phylogenetic tree.Sequence analysis data and phylogenetic tree position further demonstrate bacterial strain 140045 for Bacillusamyloliquefaciens.
Embodiment 7
The bacterial strain HMQAU140045 inhibitory action in vitro blue berry branch-rot.
Materials and methods: gather healthy blue berry branch (kind: Du Ke) raw then from blue berry planting base, Jiangnan, Qingdao, be cut into the branch section that thickness is consistent, after tap water, with 75% ethanol surface sterilization, finally use aseptic water washing.Test point four kinds sprays medicament, and (dilution of A microbial inoculum is 1 × 108The dilution of cfu/mL, B microbial inoculum, C microbial inoculum is 1 × 108Cfu/mL, D: 1500 times of diluents of thiophanate-methyl), three kinds of process (1: inoculate after dispenser 24h pathogen bacterium cake, 2: after dispenser until medicine slightly dry inoculation immediately pathogen bacterium cake, 3: dispenser after inoculation pathogen bacterium cake 24h), totally 12 kinds of combinations.
Adopting needle point method inoculation pathogen vacation cocoa hair color two born of the same parents' bacterium cake, each branch 1 point of inoculation, each process repeats 5 branches, and to inoculate the branch of sterilized water and liquid fermentation medium as comparison, test repeats 3 times.After inoculation pathogen bacterium cake, being fastened and moisturizing with preservative film on bacterium cake, condition of culture is 25 DEG C, 12h alternation of light and darkness.Remove preservative film and start observed and recorded incidence after cultivating 24h.Measure Lesion size after 7 days, calculate lesion area and prevention effect according to ellipse area formula；Prevention effect (%)=[(comparison lesion area-process lesion area)/comparison lesion area] × 100%.
Result and analysis: blue berry hair color two born of the same parents' branch-rot is all had obvious prevention effect by the bacteria suspension of bacterial strain HMQAU140045 and ferment filtrate, and is better than comparison (table 8).Result shows, inoculates in 4 process of pathogen, without significant difference between preventive effect and the preventive effect of D (thiophanate-methyl) of A (thalline) and C (fermentation liquid), it is suppressed that rate is all more than 96% after dispenser 24h；The preventive effect of B (ferment filtrate) processes less than first three, but remains to reach more than 93%.Inoculate pathogen after dispenser simultaneously, finding to there was no significant difference between B (ferment filtrate) and preventive effect and the preventive effect of D (thiophanate-methyl) of C (fermentation liquid), A (thalline) preventive effect is 82.56% significantly lower than other three kinds of process.Dispenser after inoculation pathogen 24h, find to there was no significant difference between preventive effect and the preventive effect of D (thiophanate-methyl) of C (fermentation liquid), D best results, next to that B (ferment filtrate), A (thalline) effect is worst, and bacteriostasis rate only has 67.06%.Therefore, using the fermentation liquid combination of fermentation liquid (thalline with) effect and be better than and individually use ferment filtrate and thalline, after first dispenser, inoculation pathogen effect is better than first dispenser after inoculation pathogen.
The tri-kinds of microbial inoculums of the table 8 bacterial strain HMQAU140045 prevention effect in vitro blue berry hair color two born of the same parents' branch-rot
Note: same column of figure letter representation below is at P < 0.05 level difference notable (DuncanShi duncan's new multiple range method).
Embodiment 8
The bacillus amyloliquefaciens prevention effect to blue berry hair color two born of the same parents' branch-rot.
Materials and methods: in the blue berry base of Jiangnan, Qingdao, choose health raw then and grow consistent blue berry branch, after 75% alcohol disinfecting, stab with insect needle, wound diameter is about 1-2mm, after vacation cocoa hair color two born of the same parents are activated 24 hours in PDA culture medium, beat from colony edge diameter 5mm card punch and take bacterium cake, respectively thalline (the concentration 10 of inoculating strain HMQAU1400458Cfu/mL), fermentation liquid (concentration 108Cfu/mL) and ferment filtrate is micro-dry after by pure culture biscuits involvng inoculation thereon, moisturizing 24 hours, with the branch of inoculating sterilized water and thiophanate-methyl for comparison.Measure Lesion size after 7 days and calculate prevention effect.
Result and analysis: result of the test shows, the ferment filtrate of bacterial strain HMQAU140045 and two kinds of microbial inoculums of fermentation liquid are better to the prevention effect of blue berry hair color two born of the same parents' branch-rot, and with the effect of thiophanate-methyl without significant difference (table 9).
The tri-kinds of microbial inoculums of the table 9 bacterial strain HMQAU140045 prevention effect to blue berry hair color two born of the same parents' branch-rot
Note: same column of figure letter representation below is at P < 0.05 level difference notable (DuncanShi duncan's new multiple range method).
Embodiment 9
The detection of bacterial strain HMQAU140045 inducible enzyme.
Materials and methods:
For examination culture medium:
Chitinase detection culture medium: NH4H2PO41.0g, KCl0.2g, MgSO4·7H2O0.2g, tobacco brown spot pathogen 1% (w/v) distilled water is settled to 1000mL, agar 20.0g, pH7.0；
Protease detection culture medium: casein 10.0g, beef extract powder 3.0g, NaCl5.0g, K2HPO42.0g, agar 15.0g, bromthymol blue 0.05g, pH7.4 ± 0.1；
Cellulase detection culture medium: peptone 10.0g, yeast powder 10g, sodium carboxymethyl cellulose 10.0g, NaCl5.0g, KH2PO41.0g, distilled water is settled to 1000mL, agar 18.0g, pH7.0；
β-1,3-glucanase detection culture medium: glucose 1.0g, Poria powder 4.0g, K2HPO41.0g, Na2HPO43.0g, aniline blue 0.06g, FeSO4·7H2O0.5g, agar powder 12g, distilled water is settled to 1000ml, pH7.0；
Culture medium is detected: chrome azurol S 60.5mg, 1mmol/LFeCl addicted to ferrum element3·6H2O10mL, cetyl trimethylammonium bromide 72.9mg, agar 20g, distilled water is settled to 1000mL, pH7.0.
Detecting 140045 thalline and whether ferment filtrate has enzyme and live, thalline detects: with inoculating loop picking bacterial strain 140045 thalline, point is connected on five kinds of culture medium flat plates to be measured；Ferment filtrate detects: beat, around detection flat board, the circular hole taking three diameter 6mm, adds the HMQAU140045 of the 20 μ L ferment filtrate obtained in Optimal compositions of fermentation medium and fermentation condition in circular hole.Cultivating 3d for 28 DEG C, observe chitinase, protease, β-1,3-glucanase detection culture medium flat plate, with or without transparent circle, being observed and produced with or without crocus haloing addicted to ferrum element detection culture medium flat plate, if producing transparent circle or crocus haloing, producing corresponding enzyme.Cellulase detection observational technique is the Congo red solution adding appropriate 1mg/mL in the culture dish cultivate 3d, and dye 1h, discards dye liquor, add appropriate 1MNaCl solution, washing 1h, if producing cellulase, then appearance transparent circle clearly around bacterium colony or ferment filtrate.
Result and analysis: inducible enzyme testing result finds, detect on culture medium flat plate at protease and cellulase, transparent circle (Fig. 5) can be produced around bacterial strain HMQAU140045 and ferment filtrate thereof, show that this bacterial strain can produce protease and cellulase, containing protease and cellulase in ferment filtrate, but at chitinase, β-1,3-glucanase, detect on culture medium flat plate addicted to ferrum element and do not find corresponding transparent circle or crocus haloing, show that this bacterial strain can not produce chitinase, β-1,3-glucanase and addicted to ferrum element.
Embodiment 10
The heat stability test of bacterial strain HMQAU140045 ferment filtrate.
In 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C, 121 DEG C, bacterial strain HMQAU140045 ferment filtrate is processed 60min respectively, and each process repeats 3 times.After natural cooling, with the ferment filtrate that is left intact for comparison.Each process is in triplicate.With false cocoa hair color two born of the same parents for for examination bacterium, adopt mycelial growth rate method to measure the bacteriostatic activity (ferment filtrate after process dilutes 100 times in PDA culture medium) after ferment filtrate processes at different conditions, measure each process bacteriostatic activity to pathogen respectively.
Result and 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 raises with temperature and slightly declines, but bacteriostasis rate is still higher, is closer to compareing.When height is to 100 DEG C, Antagonistic Fungi ferment filtrate bacteriostatic activity declines substantially, and bacteriostasis rate reduces 27.40% relative to comparison；When 121 DEG C, this bacterium ferment filtrate bacteriostatic activity almost total loss, this shows that in ferment filtrate, bioactive substance has stronger heat stability, but its activity can be caused and have a strong impact on by high temperature, also illustrate that its active substance such as protease and cellulase have played important function in the process of inhibition of pathogen.
The heat stability of table 10 bacterial strain HMQAU140045 ferment filtrate
Note: same column of figure letter representation below is at P < 0.05 level difference notable (DuncanShi duncan's new multiple range method)
The embodiment above is the embodiment that the present invention is best; but embodiment of the present invention are not by the restriction of the embodiment above; when other any is without prejudice to the principle of the invention, it is possible to by changing embodiment produced by the form of parameter, be all contained within protection scope of the present invention.
Nucleotide or aminoacid sequence table
Claims (10)
1. a bacillus amyloliquefaciens (Bacillusamyloliquefaciens) bacterial strain HMQAU140045, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCCNo.11768.
2. the microbial inoculum that prepared by bacillus amyloliquefaciens (Bacillusamyloliquefaciens) the bacterial strain HMQAU140045 described in claim 1.
3. microbial inoculum according to claim 2, it is characterised in that the active component of microbial inoculum is thalline and/or the ferment filtrate of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) bacterial strain HMQAU140045.
4. bacillus amyloliquefaciens (Bacillusamyloliquefaciens) the bacterial strain HMQAU140045 described in claim 1 is used for preventing and treating blue berry hair color two born of the same parents' branch-rot.
5. the microbial inoculum described in Claims 2 or 3 is used for preventing and treating blue berry hair color two born of the same parents' branch-rot.
6. the preparation method of the microbial inoculum described in a Claims 2 or 3, it is characterised in that specifically comprise the following steps that
(1) the HMQAU140045 bacterial strain of cryopreservation is activated on LB solid medium, picking list bacterium colony on LB slant medium 28 DEG C cultivate 24 hours, it is thus achieved that the strain of activation；
(2) with aseptic inoculating loop from the strain scraping a ring activation LB inclined-plane, it is inoculated in 50mLLB fluid medium 130rpm, 30 DEG C of vibration 24h, collect thalline, and to adjust cell concentration are 108Cfu/mL, it is thus achieved that seed liquor；
(3) seed liquor that step (2) obtains 1.5% is inoculated in fermentation medium by fermentation culture by volume, and in 28 DEG C, 180r/min shakes training 84h；
Shake the fermentation liquid that obtains of training centrifugal after, thalline sterilized water washes twice, centrifugal, and the thalline of scraping precipitation is placed in sterilized water, and preparation obtains the microbial inoculum A containing only thalline；
Shaking and train the fermentation liquid obtained in 4 DEG C, 12000rpm obtains supernatant in centrifugal 20 minutes, by the filter of 0.22 μm, namely obtains the microbial inoculum B containing only ferment filtrate；
Shake the fermentation liquid that obtains of training centrifugal after, the thalline of scraping precipitation is placed in ferment filtrate, obtains comprising the microbial inoculum C of thalline and ferment filtrate.
7. the preparation method of microbial inoculum according to claim 7, it is characterised in that
Consisting of of described LB solid medium and LB slant medium: tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL, pH7.0；
Consisting of of described LB fluid medium: tryptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL, pH7.0.
8. the preparation method of microbial inoculum according to claim 7, it is characterised in that consisting of of described fermentation medium: Semen Maydis powder 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 is water).
9. the method being used for detecting bacillus amyloliquefaciens (Bacillusamyloliquefaciens) bacterial strain HMQAU140045, it is characterized in that, with the genomic DNA of test strains for masterplate, with 27f (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492r (5 '-TACGGTTACCTTGTTACGACTT-3 ') for primer amplification 16SrDNA, if gained amplified production is the nucleotide sequence shown in SEQIDNo:1, it is HMQAU140045 bacterial strain.
10. the detection method of the microbial inoculum described in a Claims 2 or 3, it is characterised in that with protease and cellulase detection culture medium detection, if there being positive reaction, then for the bacterial strain HMQAU140045 microbial inoculum prepared.
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| CN106434455A (en) * | 2016-10-08 | 2017-02-22 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens with organophosphorus degrading and bacteriostatic functions and application thereof |
| CN107022494A (en) * | 2017-04-05 | 2017-08-08 | 北京环氧环保科技发展有限公司 | One plant of pine the born of the same parents bacterium BN h1 of color two and biological bacteria degradation agent and its preparation method and application |
| CN107287132A (en) * | 2017-06-02 | 2017-10-24 | 烟台出入境检验检疫局检验检疫技术中心 | One plant of photobacteria, isolation and identification method and its application |
| CN110982730A (en) * | 2019-10-23 | 2020-04-10 | 海南大学 | Microbial fertilizer, preparation method and application |
| CN111020055A (en) * | 2020-01-07 | 2020-04-17 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia gonubiensis |
| CN111041120A (en) * | 2020-01-07 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia thailandica |
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| CN112625952A (en) * | 2020-12-23 | 2021-04-09 | 天津科技大学 | Bacillus subtilis subspecies stevensis and application thereof in inhibiting cyanobacterial bloom |
| CN112852663A (en) * | 2021-01-13 | 2021-05-28 | 辽宁省农业科学院 | Bacillus amyloliquefaciens for preventing and treating peanut brown spot and application thereof |
| CN114410544A (en) * | 2022-02-18 | 2022-04-29 | 西南林业大学 | Acinetobacter cinnamoylinum HZJK-2 and application thereof |
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| CN106434455A (en) * | 2016-10-08 | 2017-02-22 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens with organophosphorus degrading and bacteriostatic functions and application thereof |
| CN106399177B (en) * | 2016-10-08 | 2019-10-22 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens and its microbial inoculum with degradation Phos and bacteriostasis |
| CN106434455B (en) * | 2016-10-08 | 2019-11-05 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens and its application with degrading organic phosphor and bacteriostasis |
| CN107022494A (en) * | 2017-04-05 | 2017-08-08 | 北京环氧环保科技发展有限公司 | One plant of pine the born of the same parents bacterium BN h1 of color two and biological bacteria degradation agent and its preparation method and application |
| CN107287132A (en) * | 2017-06-02 | 2017-10-24 | 烟台出入境检验检疫局检验检疫技术中心 | One plant of photobacteria, isolation and identification method and its application |
| CN107287132B (en) * | 2017-06-02 | 2020-07-07 | 烟台出入境检验检疫局检验检疫技术中心 | Luminescent bacterium, separation and identification method and application thereof |
| CN110982730A (en) * | 2019-10-23 | 2020-04-10 | 海南大学 | Microbial fertilizer, preparation method and application |
| CN111041120A (en) * | 2020-01-07 | 2020-04-21 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia thailandica |
| CN111020055A (en) * | 2020-01-07 | 2020-04-17 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia gonubiensis |
| CN111020055B (en) * | 2020-01-07 | 2022-07-08 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia gonubiensis |
| CN111041120B (en) * | 2020-01-07 | 2022-07-08 | 北京林业大学 | LAMP primer and kit for detecting Lasiodipia thailandica |
| CN112359001A (en) * | 2020-11-27 | 2021-02-12 | 巴州加木农业科技有限公司 | Bacillus amyloliquefaciens microbial agent and application thereof |
| CN112359001B (en) * | 2020-11-27 | 2022-11-29 | 巴州加木农业科技有限公司 | Bacillus amyloliquefaciens microbial agent and application thereof |
| CN112625952A (en) * | 2020-12-23 | 2021-04-09 | 天津科技大学 | Bacillus subtilis subspecies stevensis and application thereof in inhibiting cyanobacterial bloom |
| CN112625952B (en) * | 2020-12-23 | 2022-05-06 | 天津科技大学 | Bacillus subtilis subspecies schoenoprasu and application thereof in inhibiting cyanobacterial bloom |
| CN112852663A (en) * | 2021-01-13 | 2021-05-28 | 辽宁省农业科学院 | Bacillus amyloliquefaciens for preventing and treating peanut brown spot and application thereof |
| CN114410544A (en) * | 2022-02-18 | 2022-04-29 | 西南林业大学 | Acinetobacter cinnamoylinum HZJK-2 and application thereof |
| CN114410544B (en) * | 2022-02-18 | 2023-06-23 | 西南林业大学 | Acinetobacter cinnarii HZJK-2 and application thereof |
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