CN109929782A - It is a kind of inhibit Phomopsis bacillus subtilis and its application - Google Patents
It is a kind of inhibit Phomopsis bacillus subtilis and its application Download PDFInfo
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Abstract
The present invention provides a kind of bacillus subtilis for inhibiting Phomopsis, the bacillus subtilis is bacillus subtilis (Bacillus subtilis) T8, China typical culture collection center, deposit number are preserved on December 05th, 2018 are as follows: CCTCC NO:M 2018860.Bacillus subtilis T8 provided by the invention has good cellulose degradation ability, can generate cellulase, and has stronger Endoglucanases activity, Filter paperlyase activity, 5 prime excision enzyme activity and activity of beta-glucosidase.Bacillus subtilis T8 provided by the invention is able to suppress Phomopsis and its ethyl alcohol supernatant protein all has inhibitory effect to Phomopsis.In addition, bacillus subtilis T8 is able to suppress the growth of Escherichia coli, staphylococcus aureus, Candida albicans, shigella dysenteriae and excrement enterobacteria, there is value of exploiting and utilizing in inhibiting human disease bacterium.The present invention also provides a kind of bacillus subtilis T8 to produce cellulase and prevent and treat the application in Kiwi berry soft rot.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to it is a kind of inhibit Phomopsis bacillus subtilis and its answer
With.
Background technique
Phomopsis (Phomopsis) is Fungi Imperfecti door, Coelomycetes, Sphaeropsidales, one in sphereioidaceae mycosis
It is big to belong to, containing 100 multiple and different kinds, more than 70 kinds of not equal plants can be parasitized.The category pathogen Regional Distribution is extensive, draws
The serious plant diseases such as leaf withered, branch is withered, rotten stem, ulcer and the fruit corruption of plant are played, are resulted in significant economic losses.As it is that Kiwi berry is adopted
Soft rot after plucking.Kiwi berry branch-rot pathogen, peach deadlock bud, the pathogenic strain of plantain head blight.
Prevention and treatment Phomopsis mainly uses chemical pesticide at present, such as: Yang Tingmi intends stem point to citrus using 8 kinds of pesticides
(the .8 kind pesticide such as Yang Tingmi is to Phomopsis citri toxicity test and field controling test for mould indoor virulence and field control
Southern china fruit tree .2014,43 (2): 52-53).The interior that Sun Yanfang etc. carries out Asparagus Stem Blight using 15 kinds of chemical pesticides
Screening experiment (toxicity test .2013 of the .15 kind medicament such as Sun Yanfang to Asparagus Stem Blight pathogen, 33 (4): 71-75), Wu Wen
More than 20 fungicide can be tested to indoor virulence (the Kiwi berry soft rot nosophyte such as Wu Wenneng of Kiwi berry main pathogenic bacteria
Separation identification and antibacterial agent screening plant protection journal .2017.44 (5): 826-832), but microbial control agent report compared with
It is few.
Summary of the invention
It is an object of that present invention to provide a kind of bacillus subtilis for inhibiting Phomopsis and its applications.
To achieve the above object, the present invention provides a kind of bacillus subtilis for inhibiting Phomopsis, the withered grass gemma
Bacillus is bacillus subtilis (Bacillus subtilis) T8, is preserved in Chinese Typical Representative culture on December 05th, 2018
Collection, deposit number are as follows: CCTCC NO:M 2018860.
Preferably, the 16s rDNA of bacillus subtilis (Bacillus subtilis) T8 has such as SEQ ID:1
Shown in nucleotide sequence.
Preferably, bacillus subtilis (Bacillus subtilis) T8 can inhibit Escherichia coli, golden yellow grape
Coccus, Candida albicans, shigella dysenteriae and excrement enterobacteria.
Preferably, being inoculated in the bacillus subtilis (Bacillus subtilis) T8 with sodium carboxymethylcellulose
(CMC-Na) cellulase is made on the enzymatic production culture medium of substrate, to carry out fermented and cultured.
Preferably, the enzymatic production culture medium includes 8~15g/L of CMC-Na, (NH4)2SO42~5g/L, MgSO4·
7H20.3~0.7g/L of O, K2HPO41~3g/L, 2~6g/L of beef extract, 6~12g/L of peptone.
Preferably, the fermentation culture conditions are, cultivation temperature is 23~35 DEG C, and shaking speed is 120~180rpm/
min.The present invention also provides a kind of application of the bacillus subtilis of above-mentioned inhibition Phomopsis in prevention and treatment Kiwi berry soft rot.
Bacillus subtilis T8 provided by the invention has the advantage that
(1) bacillus subtilis T8 provided by the invention has good cellulose degradation ability, can generate cellulose
Enzyme, and there is stronger Endoglucanases activity, Filter paperlyase activity, 5 prime excision enzyme activity and activity of beta-glucosidase,
Producing has application value on cellulase.
(2) bacillus subtilis T8 provided by the invention is able to suppress Phomopsis and its ethyl alcohol supernatant protein pair
Phomopsis all has inhibitory effect, has application value in prevention and treatment Kiwi berry soft rot.In addition, bacillus subtilis T8 energy
The growth for enough inhibiting Escherichia coli, staphylococcus aureus, Candida albicans, shigella dysenteriae and excrement enterobacteria is inhibiting the mankind
There is value of exploiting and utilizing in pathogenic bacteria.
Bacillus subtilis T8 of the invention, the deposit date is on December 05th, 2018, deposit number was CCTCC NO:M
2018860, classification naming is bacillus subtilis (Bacillus subtilis), depositary institution's title are as follows: Chinese Typical Representative culture
Object collection, collection address are as follows: Wuhan, China Wuhan University.
Detailed description of the invention
Fig. 1 is bacillus subtilis hydrolysis figure in embodiment 1；
Fig. 2 is glucose standard curve in embodiment 2；
Fig. 3 is Phomopsis bacteriostatic experiment result figure in embodiment 3；
Fig. 4 is bacillus subtilis T8 protein and ethyl alcohol supernatant Phomopsis bacteriostatic experiment result figure in embodiment 3；
Fig. 5 is Escherichia coli bacteriostatic experiment result figure in embodiment 3；
Fig. 6 is staphylococcus aureus bacteriostatic experiment result figure in embodiment 3；
Fig. 7 is Candida albicans bacteriostatic experiment result figure in embodiment 3；
Fig. 8 is shigella dysenteriae bacteriostatic experiment result figure in embodiment 3；
Fig. 9 is excrement enterobacteria bacteriostatic experiment result figure in embodiment 3.
Specific embodiment
Purpose in order to better illustrate the present invention, technical scheme and beneficial effects, below in conjunction with attached drawing and specific implementation
The invention will be further described for example.It should be noted that following the methods of implementing are explained further to what the present invention was done
It is bright, it should not be taken as limitation of the present invention.If material used in the embodiment of the present invention, reagent all can be from without specified otherwise
Commercial sources obtain.
The screening of 1 cellulase producing strain of embodiment
Screening and culturing medium: CMC-Na 10g/L, (NH4)2SO4 1.4g/L、MgSO4 0.3g/L、KH2PO42g/L、MnSO4
1.6mg/L、FeSO4 5mg/L、ZnSO4 2.5mg/L、CoCl22.0mg/L, agar 20g/L, pH 7.0
Seed culture medium: CMC-Na 10g/L, peptone 3g/L, KH2PO4 4g/L、MgSO4·7H2O 0.03g/L、pH
6.0。
Enzymatic production culture medium: CMC-Na 10g/L, (NH4)2SO4 4.0g/L、MgSO4·7H2O 0.5g/L、K2HPO4
2g/L, beef extract 5g/L, peptone 10g/L (pH is natural)
Primary dcreening operation: Nanchang Qinshan Lake bottom mud soil sample 10g is weighed, is put into the triangular flask equipped with 90mL sterile water, sets
It is taken out after shaking table 150rpm vibrates 30min, is diluted to 10 step by step-1, 10-2, 10-3, 10-4, 10-5, 10-66 kinds of concentration, by 6
The dilution of kind concentration is respectively coated on screening and culturing medium, is repeated 3 times, in 25 DEG C of culture 3d. after mycelia grows, is inoculated
It is purified on new screening and culturing medium, until obtaining pure bacterial strain.
Secondary screening: transparent circle method primary dcreening operation is known as the bacterial strain of degrading activity to fiber, by the single bacterial strain and its Mixed Microbes of separation
Dibbling is on screening and culturing medium respectively, and 25 DEG C of culture 3d, with 1.0g/L congo red staining 30min, incline dye liquor, then uses 1mol/L
NaCl aqueous solution decolourize 1h, survey colony diameter (d, cm) and hydrolytic circle (D, cm), hydrolysis ability: Dp indicated using Dp
=(D/d)2。
The results show that bacterial strain T8 has good cellulose degradation ability, as shown in Figure 1, Dp=(D/d)2=23.Bacterial strain
T8 is accredited as bacillus subtilis (Bacillus subtilis), and 16s rDNA has the nucleotide as shown in SEQ ID:1
Sequence.
The measurement of 2 bacillus subtilis T8 degrading enzymatic activity of embodiment
Using 3,5- dinitrosalicylic Acid Colorimetry (DNS method) measurement Filter paperlyase activity, endo-type glucosidase activity,
5 prime excision enzyme activity and activity of beta-glucosidase.
The production of 2.1 glucose standard curves
Glucose standards solution: the DEXTROSE ANHYDROUS 1g that drying to constant weight at 103 DEG C is weighed, is settled to 100mL with water；
Phosphate buffer: 0.1mol/L pH 6.0 (being suitable for neutral cellulase) weighs sodium dihydrogen phosphate-water respectively
121.0g and phosphate dihydrate disodium hydrogen 21.89g, is dissolved in 1L deionized water.Adjust the pH to 6.0 of solution；
DNS reagent: weighing 3,5- dinitrosalicylic acid 10g, is placed in about 600mL water, is gradually added into sodium hydroxide l0g,
In 50 DEG C of water-baths after (magnetic force) stirring and dissolving, sodium potassium tartrate tetrahydrate 200g is sequentially added, phenol (steams) 2g and anhydrous sulfurous again
Sour sodium 5g is cooled to room temperature after all dissolving and clarifying, is settled to 1000mL with water, filters.It is stored in brown reagent bottle
In, it is used after 7d is placed in dark place.
Glucose standards stock solution 0.0,1.0,1.5,2.0,2.5,3.0,3.5mL are drawn respectively in l0mL, volumetric flask
In, it is settled to 10mL, Gai Sai with water, is shaken up spare.
It is measured by as defined in table 1, it is (every in each pipe using solution, buffer solution and DNS reagent to draw Glucose standards respectively
Pipe number makees 3 samples in parallel), it mixes.Standard pipe is placed in boiling water bath simultaneously, reacts 10min.It takes out, is rapidly cooled to room temperature,
It is settled to 25mL, Gai Sai with water, is mixed.With 10mm cuvette, absorbance is measured at spectrophotometer wavelength 540nm.With Portugal
Grape sugar amount is abscissa, using absorbance as ordinate, draws standard curve, obtains equation of linear regression, linear regression coeffficient is answered
It can use and (otherwise must reform) at 0.9990 or more.
1 glucose standard curve of table
Gained glucose standard curve is as shown in Fig. 2, y=0.334x-0.0184.
2.2DNS method measures cellulase
Sodium cellulose glycolate (CMC Na) chemistry pure (Shanghai brilliance chemical reagent factory) is at 25 DEG C, 2% aqueous solution, viscosity
800mPa·s-1200mPa·s。
CMC-Na solution: weighing 2g CMCNa, be accurate to 1mg, is slowly added into phosphate buffer 200mL and is heated to 80
DEG C -90 DEG C, the side Bian Jiare magnetic agitation with corresponding buffer is diluted to 300mL after cooling until CMC-Na all dissolves,
With the pH of 2mol/L hydrochloric acid or sodium hydrate regulator solution to (6.0 scholar 0.05) (neutral cellulase), last constant volume is arrived
300mL is stirred evenly, and is stored in spare in refrigerator.
Crude enzyme liquid preparation: the bacterial strain that primary dcreening operation obtains is inoculated in enzymatic production culture medium respectively by 5% inoculum concentration,
After (25 DEG C, 150rpm/min) culture 3d of shaking table, 8000 × g is centrifuged 10min, and taking supernatant is the crude enzyme liquid prepared.
(1) endo-type glucuroide (endo-1,4- β-D-glucanase, EC3.3.1.4, abbreviation EBG), methylol
Cellulose (reducing sugar method) enzyme activity (CMCA-DNS) measuring method
Four 25mL scale tool plug test tubes (blank tube, three sample cells) are taken, it is accurate to be added respectively into four branch pipes
With the CMC-Na solution 2.00mL of corresponding pH buffer preparation, the crude enzyme liquid 0.50mL Yu Sanzhi diluted is accurately added in difference
In sample cell (blank tube is not added), mixed with eddy blending machine, Gai Sai.Four test tubes are placed in (50+0.1) DEG C water-bath simultaneously
In, 30min is reacted in accurate timing, takes out.DNS reagent 3.0mL is quickly and accurately added into each pipe, it is accurate in blank tube
The enzyme solution 0.50mL to be measured diluted is added, shakes up.Four branch pipes are put into boiling water bath simultaneously, 10min is heated in accurate timing,
It takes out, is rapidly cooled to room temperature, is settled to 25mL with water.With blank tube (comparison liquid) demodulating apparatus zero point, in spectrophotometer wave
Under long 540nm, with 10mm cuvette, the absorbance of sample liquid in three sample cells is measured respectively, is averaged.By looking into standard song
Line or the content that reduced sugar is found out with equation of linear regression.
CMCA-DNS enzyme activity, is calculated as follows.
In formula: X1-hydroxymethyl cellulose (reducing sugar method) enzyme activity (CMCA-DNS), u/g (or u/mL)；
A is-absorbance checks in the reduction sugar amount of (or calculating), mg on standard curve；
1/0.5 be-is converted into enzyme solution 1mL；
N is the-extension rate of enzyme sample；
2 be-time scale factor.
(2) measuring method of filter paper enzyme activity (FPA):
Diameter 15cm fast qualitative filter paper (Hangzhou Xinhua No.1 filter paper) filter paper is put into (silica gel) drier and is balanced
24h；By the filter paper item that wide 1cm is made in the filter paper after water balance, quality is 50mg, it is converted into M type, it is spare.Four 25mL are taken to carve
It spends tool plug test tube (blank tube, three sample cells).It will be converted into the filter paper item of M type, is respectively put into the bottom (edge of every test tube
The direction lcm is put into vertically).Respectively into four branch pipes, the buffer solution 1.50mL of corresponding pH is accurately added.It is accurately added respectively dilute
In the enzyme solution 0.50mL Yu Sanzhi sample cell to be measured released (blank tube is not added), solution in pipe is made to submerge filter paper, Gai Sai.By four
Test tube is placed in simultaneously in (50 scholar 0.1) DEG C water-bath, accurate timing, reacts 60min, is taken out.It is accurately added immediately into each pipe
DNS reagent 3.0mL.The enzyme solution 0.50mL to be measured diluted is accurately added in blank tube, shakes up.Four branch pipes are put into simultaneously
In boiling water bath, 10min is heated, is taken out, is rapidly cooled to room temperature, water is added to be settled to 25mL, shake up.With blank tube (comparison liquid) tune
Instrument zero, with 10mm cuvette, measures the extinction of sample liquid in three parallel pipes at spectrophotometer wavelength 540nm respectively
Degree, is averaged.Standard curve is looked into absorbance values or the content of reduced sugar is found out with equation of linear regression.
By filter paper enzyme activity (FPA), formula following formula is calculated.
X1The filter paper enzyme activity (FPA) of-sample, μ/g (or μ/mL)；
A-checks in the reduction sugar amount of (or calculating), mg according to absorbance on standard curve；
1/0.5-is converted into enzyme solution 1mL；
N-enzyme sample extension rate.
(3) 5 prime excision enzyme activity measures
Method only replaces filter paper with absorbent cotton with (2)
(4) measurement of beta-glucosidase (β-Isosorbide-5-Nitrae-glucosidase, β-Gase) vigor
Method only substitutes CMC-Na solution with salicin solution with (1).
According to y=0.334x-0.0184, the glucose content in each anti-liquid is calculated, calculates Filter paperlyase further according to formula
Activity and endo-type glucosidase activity, 5 prime excision enzyme activity and activity of beta-glucosidase the results are shown in Table 2:
2 DNS method of table measures cellulase activity
| Number | Endoglucanases enzyme activity | Filter paperlyase enzyme activity | Excision enzyme enzyme activity | B-Gase |
| 8 | 53.34u/mL | 18.34u/mL | 19.6u/mL | 32u/mL |
3 bacillus subtilis T8 bacteriostatic experiment of embodiment
Enzymatic production culture medium: CMC-Na 10g, (NH4)2SO4 4.0g、MgSO4·7H2O 0.5g、K2HPO42g, ox
Meat extract 5g, peptone 10g (pH nature constant volume to 1L)
LB liquid medium: tryptone (Tryptone) 10g/L, yeast extract (Yeast-extract) 5g/L, chlorine
Change sodium (NaCl) 10g/L, is adjusted to pH 7.0.
LB solid medium: tryptone (Tryptone) 10g/L, yeast extract (Yeast-extract) 5g/L, chlorine
Change sodium (NaCl) 10g/L, agar 20g/L and is adjusted to pH 7.0.
PDA culture medium: dehydrated potato powder 6.0g/L, glucose 20.0g/L, agar 20.0g/L, pH value 5.4-5.8
3.1 Phomopsis
Phomopsis is coated on PDA plate, 37 DEG C, after cultivating 7d, washes lower spore with physiology salt, Phomopsis is quasi- stem
The suspension of the mould spore of point.
Bacillus subtilis T8 bacterium solution is inoculated in the test tube equipped with 5mL enzymatic production culture medium by 5% inoculum concentration, and 25
DEG C, 150rpm/min cultivates 2d, takes 500 μ L bacteria suspensions into 1.5mL centrifuge tube, and 8000 × g is centrifuged 5min, and take supernatant spare,
The as antibacterial stoste of bacillus subtilis T8.Positive control are as follows: the dual anti-(content of penicillin of mycillin mixed liquor (100 ×)
For 10kU/mL, the content of streptomysin is 10mg/mL.)
40 μ L Phomopsis spore suspensions are taken to be coated on LB plate, the diameter 1.4cm circle Xinhua filter paper of sterilizing after 2h
It is affixed on plate, takes the antibacterial stoste of 10 μ L bacillus subtilis T8 and 10 μ L mycillin mixed liquor (100 ×) dual anti-drops respectively
It is added on filter paper, 37 DEG C, cultivates 1d, measure antibacterial circle diameter ratio, calculate antibacterial circle diameter ratio and round Xinhua filter paper diameter ratio.
As a result as shown in figure 3, in figure → instruction be bacillus subtilis T8 inhibition zone,What is indicated is control
Inhibition zone, bacillus subtilis T8 is 1.8 to the diameter ratio of Phomopsis, and compareing dual anti-diameter ratio is 1.
0.4mL fermentation liquid is separately taken, 0.4mL dehydrated alcohol is added, centrifugation is water-soluble with 100 μ L after taking supernatant, precipitating dry
Solution takes 10 μ L to carry out ethyl alcohol supernatant albumen bacteriostatic experiment respectively.
As a result as shown in figure 4, in figure → instruction is protein,What is indicated is ethyl alcohol supernatant, bacillus subtilis
The ethyl alcohol supernatant protein of T8 is all effective to Phomopsis.
3.2 Escherichia coli, staphylococcus aureus, Candida albicans, shigella dysenteriae and excrement enterobacteria
Escherichia coli, staphylococcus aureus, Candida albicans, shigella dysenteriae and excrement enterobacteria single colonie are inoculated with respectively
Amount is inoculated in the test tube equipped with 5mL LB culture liquid base, and 37 DEG C, 150rpm/min, culture 20h is respective bacteria suspension.
Other steps are the same as 3.1.
As a result as shown in figures 5-9, Fig. 5 is Escherichia coli, and Fig. 6 is staphylococcus aureus, and Fig. 7 is Candida albicans, Fig. 8
For shigella dysenteriae, Fig. 9 is excrement enterobacteria, in figure → instruction be bacillus subtilis T8 inhibition zone,What is indicated is pair
According to inhibition zone.The antibacterial circle diameter measured is than as shown in table 3.
3 bacillus subtilis T8 bacteriostatic experiment of table
| Escherichia coli | Staphylococcus aureus | Candida albicans | Shigella dysenteriae | Excrement enterobacteria | |
| T8 | 1.4 | 1.29 | 1.38 | 2.5 | 1.33 |
| It is dual anti- | 2.69 | 1.92 | 2.14 | 2.14 | 1.42 |
Prevention and treatment of the 4 bacillus subtilis T8 fermentation liquid of embodiment to Kiwi berry soft rot after harvesting
Bacillus subtilis T8 bacterium solution is inoculated in the fermentor equipped with 5L enzymatic production culture medium by 5% inoculum concentration,
25 DEG C, 150rpm/min cultivates 2d, and fermentation liquid stands 1d, takes supernatant, then 8000 × g to be centrifuged 10min, takes supernatant spare, as
Bacillus subtilis T8 prevents and treats liquid.
The Kiwi berry that bacillus subtilis T8 prevention and treatment liquid even spraying is newly picked in 100, dries, room temperature is stored up naturally
Hiding.Meanwhile room temperature stores 100 Kiwi berrys newly picked naturally, bacillus subtilis T8 prevention and treatment liquid is not sprayed, as control.15d
After calculate respective soft rot incidence.
The results show that after the Kiwi berry room temperature for having sprayed bacillus subtilis T8 prevention and treatment liquid stores 15d naturally, wherein mature
Kiwi berry, soft rot incidence be 1.5%, control group do not sprayed bacillus subtilis T8 prevention and treatment liquid Kiwi berry store naturally
After 15d, wherein mature Kiwi berry, soft rot incidence is 65%.It can be seen that bacillus subtilis T8 prevention and treatment liquid can be significant
Reduce the incidence of Kiwi berry soft rot after harvesting.
Bacillus subtilis T8 provided by the invention has good cellulose degradation ability, can generate cellulase,
And there is stronger Endoglucanases activity, Filter paperlyase activity, 5 prime excision enzyme activity and activity of beta-glucosidase, it is producing
There is application value on cellulase.
Bacillus subtilis T8 provided by the invention is able to suppress Phomopsis and its ethyl alcohol supernatant protein to quasi- stem
Point is mould to all have inhibitory effect, has application value in prevention and treatment Kiwi berry soft rot.In addition, bacillus subtilis T8 can press down
The growth of Escherichia coli, staphylococcus aureus, Candida albicans, shigella dysenteriae and excrement enterobacteria processed is inhibiting human disease
There is value of exploiting and utilizing in bacterium.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<110>institute of microbiology, Jiangxi Prov. Academy of Science
<120>a kind of bacillus subtilis for inhibiting Phomopsis and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1463
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 1
acttctgtca cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca 60
aactctcgtg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg 120
ctgatccgcg attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga 180
actgagaaca gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc 240
cattgtagca cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac 300
cttcctccgg tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa 360
gmtccaaagg agttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420
gacaaccatg caccacctgt cactctgccc ccgaagggga cgtcctatct ctaggattgt 480
cagaggatgt caagacctgg taagggttct tcgcgttgct tcgaattaaa ccacatgctc 540
caccgcttgt gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc 600
aggcggagtg cttaatgcgt tagctgcagc actaaggggc ggaaaccccc taacacttag 660
cactcatcgt ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt 720
cgctcctcag cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat 780
ctctacgcat ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc 840
cagtttccaa tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg 900
cctgcgagcc ctttacgccc aataattccg gacaacgctt gccacctacg tattaccgcg 960
gctgctggca cgtagttagc cgtggctttc tggttaggta ccgtcaaggt accgccctat 1020
tcgaacgggt acttgttctt ccctaacaac agagctttga cgatccgaaa accttsgatc 1080
actcacgcgg gcgtttgctc cgtcagaact ttcgtccatt gcggaagatt ccctactgct 1140
gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt 1200
cggctacgca tcgttgcctt ggtgagccgt tacctcacca actagctaat gcgccgcggg 1260
tccatctgta agtggtagcc gaagccacct tttatgtttg aaccatgcgg ttcaaacaac 1320
catccggtat tagccccggt ttcccggagt tatcccagtc ttacaggcag gttacccacg 1380
tgttactcac ccgtccgccg ctaacatcag ggagcaagct cccatctgtc cgctcgactg 1440
catgtatagc acccgccacg gcc 1463
Claims (7)
1. a kind of bacillus subtilis for inhibiting Phomopsis, it is characterised in that: the bacillus subtilis is bacillus subtilis
Bacterium (Bacillus subtilis) T8 is preserved in China typical culture collection center, deposit number on December 05th, 2018
Are as follows: CCTCC NO:M 2018860.
2. inhibiting the bacillus subtilis of Phomopsis as described in claim 1, it is characterised in that: the bacillus subtilis
The 16s rDNA of (Bacillus subtilis) T8 has the nucleotide sequence as shown in SEQ ID:1.
3. inhibiting the bacillus subtilis of Phomopsis as described in claim 1, it is characterised in that: the bacillus subtilis
(Bacillus subtilis) T8 can inhibit Escherichia coli, staphylococcus aureus, Candida albicans, shigella dysenteriae and excrement intestines
Bacillus.
4. the bacillus subtilis as described in any one of claims 1-3 for inhibiting Phomopsis is producing the application in cellulase,
It is characterized by: being inoculated in the bacillus subtilis (Bacillus subtilis) T8 with sodium carboxymethylcellulose (CMC-
Na cellulase) is made on the enzymatic production culture medium of substrate, to carry out fermented and cultured.
5. inhibiting application of the bacillus subtilis of Phomopsis in production cellulase as claimed in claim 4, feature exists
In: the enzymatic production culture medium includes 8~15g/L of CMC-Na, (NH4)2SO42~5g/L, MgSO4·7H2O 0.3~
0.7g/L、K2HPO41~3g/L, 2~6g/L of beef extract, 6~12g/L of peptone.
6. inhibiting application of the bacillus subtilis of Phomopsis in production cellulase as claimed in claim 4, feature exists
In: the fermentation culture conditions are that cultivation temperature is 23~35 DEG C, and shaking speed is 120~180rpm/min.
7. the bacillus subtilis as described in any one of claims 1-3 for inhibiting Phomopsis is in prevention and treatment Kiwi berry soft rot
Application.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910297383.6A CN109929782B (en) | 2019-04-12 | 2019-04-12 | Bacillus subtilis for inhibiting phomopsis and application thereof |
Applications Claiming Priority (1)
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