CN109749974A - A kind of Bacillus amyloliquefaciens strain and its application - Google Patents
A kind of Bacillus amyloliquefaciens strain and its application Download PDFInfo
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Abstract
The invention discloses a kind of Bacillus amyloliquefaciens strain and its applications.The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YB1701 is CGMCC No.16003 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Bacillus amyloliquefaciens YB1701 provided by the present invention is from the fluffy rhizosphere soil of alkali, it can effective 16 kinds of plant pathogenic fungis such as antagonism Black bundle disease of maize bacterium (Acremonium strictum), yellow fusarium germ (Fusarium culmorum), phytophthora capsici (Phytophthora capsici Leonian), Melon fusarium Wilt (Fusarium oxysporum f.sp.melonis), effect is remarkably promoted to young rice seedlings growth, is had broad application prospects in biological pesticide and biological organic fertilizer development.
Description
Technical field
The invention belongs to bacterial strain Cultivating techniques fields, and in particular to a kind of Bacillus amyloliquefaciens strain YB1701 and its answer
With specially one kind can produce acc deaminase, IAA auxin and the Xie Dian to plant pathogenic fungi with wide spectrum antagonism
Afnyloliquefaciens and application thereof, can 6 kinds of frequently seen plants disease fungus of antagonistic 11, and to rice seedling have obvious growth promoting function
Bacterium.
Background technique
Alkali fluffy (Suaeda glauca Bunge) also known as alkali wormwood artemisia, salt wormwood artemisia, are born in seabeach, river valley, roadside, field more
On equal salt-soda soils, belong to Li Ke Suaeda, be a kind of extremely strong halophytes of salt tolerance, in provinces and regions distributions such as China is new, illiteracys, the Liao Dynasty
Extensively.The fluffy preferred plant variety for having become biological modification salinized soil of alkali at present.Research shows that containing rich in root system of plant soil
Rich microorganism facilitates plant absorption nutrient, promotes plant growth.Yuan Ye (2018) is from the fluffy root soil of Panjin Honghe fault alkali
It is middle to utilize isolated 80 plants thermophilic saline and alkaline bacteriums of culture-dependent method.
Bacillus amyloliquefaciens are a kind of bacteriums (Wu Yijing etc., 2012) with broad-spectrum antibacterial activation characteristics, can be produced
Raw a large amount of secondary metabolites and a variety of antibacterial substances.Qiu Sixin etc. (2004) isolates one plant to Filamentous true from tobacco stalk
Bacterium has the interior raw bacillus amyloliquefaciens TB2 of fine inhibiting effect；Arrebola etc. (2009) is isolated from fruit surface to 7
Plant the bacillus amyloliquefaciens PPCB004 that the fungal pathogens after different citruses are adopted have antibacterial activity；Wong etc. (2010) from
The Antagonistic protein that there is inhibitory activity to a variety of germs is isolated in bacillus amyloliquefaciens NK10.Zhang Shuyan etc. (2017) separation
There are the bacillus amyloliquefaciens for significantly inhibiting effect in the prevention and treatment of numb Chinese yam root rot for one plant out, bacteriostasis rate reaches
89.6%, it is significantly higher than the processing of application chemical agent fenaminosulf wettable powder.Li Shezeng etc. (2017) isolates one plant and prevents
The solution starch bud pole bacterium for controlling graw mold of tomato, can prevent and treat by the microbial fruit rot of Botrytis cinerea, leaf, stem and spend rotten
Disease, tomato production promoting can be made.
Microbial manure plays a significant role in agricultural production, and wherein strain is microbial manure production using base
Plinth.One side microorganism can generate metabolite that is active, promoting plant growth；On the other hand, microorganism can
Antagonistic phytopathogen improves crop resistance etc..
Summary of the invention
The present invention provides a kind of Bacillus amyloliquefaciens strain YB1701 and its application, has broad-spectrum antibacterial effect, can
Generate the bacillus amyloliquefaciens of acc deaminase and IAA auxin, great Development volue.
The invention is realized in this way providing a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
Bacterial strain, the Bacillus amyloliquefaciens strain is YB1701, and the Bacillus amyloliquefaciens strain YB1701 is preserved in China
Microbiological Culture Collection conservator's common micro-organisms center, deposit number are CGMCC No.16003.
A kind of microbial inoculum is provided, the active constituent of the microbial inoculum is above-mentioned bacillus amyloliquefaciens YB1701 or solution starch gemma
The fermentation liquid of bacillus YB1701 or the metabolite of bacillus amyloliquefaciens YB1701.
Above-mentioned microbial inoculum is following any microbial inoculums:
1) inhibit the microbial inoculum of plant pathogenic fungi；
2) microbial inoculum of plant pathogenic fungi disease is prevented and treated；
3) promote the microbial inoculum of plant growth；
4) microbial inoculum of stress resistance of plant is improved；
5) plant is reduced to the microbial inoculum of adverse circumstance sensibility；
6) microbial inoculum of 1-Aminocyclopropane-1-carboxylate deaminase (ACC) is generated；
7) microbial inoculum of indole-3-acetic acid (IAA) is generated；
The adverse circumstance is arid, high temperature, pest and disease damage invasion or heavy metal pollution.
Above-mentioned plant pathogenic fungi is following at least one plant pathogenic fungi:
1) Black bundle disease of maize bacterium (Acremonium strictum)；
2) Exserohilum turcicum (Exserohilum turcicum)；
3) Pythium aphanidermatum (Pythium aphanidermatum)；
4) corn rotten mildew fungus (Pythium gramincola Subra)；
5) D. carbonum (Bipolaris zeicola)；
6) phytophthora capsici (Phytophthora capsici Leonian)；
7) botrytis cinerea (Botrytis cinerea Pers.)；
8) Apple Mould Core bacterium (Trichothecium roseum))；
9) Pyricularia oryzae (Pyricularia grisea)；
10) Melon fusarium Wilt (Fusarium oxysporum f.sp.melonis)；
11) target leaf spot of sorghum bacterium (Bipolaris sorghicola)；
12) Maize Curvularia (Curvularia lunata)；
13) stenocarpella maydis (Fusarium graminearum)；
14) yellow fusarium germ (Fusarium culmorum)；
15) corn top-rotten disease bacterium (Frumentum subglutinans)；
16) sunflower kernel cup fungi (Sclerotinia sclerotorium).
A kind of biological organic fertilizer is provided, the active constituent of the biological organic fertilizer is solution starch bud described in claim 1
The fermentation liquid of spore bacillus YB1701 or bacillus amyloliquefaciens YB1701 or the metabolite of bacillus amyloliquefaciens YB1701.
A kind of cultural method of above-mentioned Bacillus amyloliquefaciens strain is provided, including bacillus amyloliquefaciens YB1701 is existed
The step of being cultivated in culture medium for cultivating bacillus amyloliquefaciens.
Specifically, the above method includes the following steps:
1) it takes the rhizosphere of fresh acquisition to have the fluffy grass of alkali of soil, shakes off its foundation soil, and weigh 10g foundation soil；
2) after foundation soil air-dries, pedotheque is placed in bead and the taper of 90mL sterile saline is housed
In bottle, room temperature 120rpmin vibrates 30min, and soil supension is made；
3) it takes 1mL soil supension to carry out gradient dilution into the test tube that 9mL sterile saline is housed, takes 10 respectively-3,
10-4, 10-5Dilution 0.1mL is applied on screening and culturing medium plate, and for 24 hours, picking single colonie is isolated and purified for 37 DEG C of cultures,
Obtain bacillus amyloliquefaciens YB1701.
Above-mentioned screening and culturing based component are as follows: beef extract 3g, peptone 10g, NaCl 5g, agar powder 18-20g, water
1000mL, pH 7.2-7.4,121 DEG C of sterilizing 20min.
Specifically, the preparation of the fermentation liquid of bacillus amyloliquefaciens YB1701 includes the following steps:
1) by the bacillus amyloliquefaciens YB1701 of purifying culture by inclined plane inoculating to beef extract-peptone solid medium
In, it is cultivated for 24 hours at 37 DEG C, 180r/min；
2) bacterial strain by step 1) activation accesses in LB liquid medium, the shaken cultivation in 37 DEG C, 180r/min shaking table
48h obtains seed liquor；
3) the resulting seed liquor of step 2) is inoculated in beef-protein medium according to 5% inoculum concentration, in 37 DEG C
Shaken cultivation 48h in 180r/min shaking table obtains bacillus amyloliquefaciens YB1701 fermentation liquid.
In the fermentation liquid of the bacillus amyloliquefaciens YB1701 bacillus amyloliquefaciens YB1701 thallus content be 1.0 ×
106~1.0 × 108cfu/mL。
Bacterial strain preservation of the present invention is described as follows:
Strain name: bacillus amyloliquefaciens
Latin name: Bacillus amyloliquefaciens
Strain number: CGMCC No.16003
Preservation mechanism: in Chinese microorganism strain preservation conservator's common micro-organisms center
Preservation mechanism abbreviation: CGMC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on June 25th, 2018
Collection is registered on the books number: CGMCC No.16003
Compared with the prior art, the advantages of the present invention are as follows: bacillus amyloliquefaciens provided by the invention
(B.amyloliquefaciens) YB1701 is separated from the fluffy rhizosphere soil of alkali, which can produce 1- amino-cyclopropane-
1- carboxylic acid (ACC) deaminase and IAA auxin have obvious bacteriostasis to for 16 kinds of plant pathogenic fungis of examination, in phytopathy
Have broad application prospects in terms of fungus diseases biological control and in terms of growth promotion.
Detailed description of the invention
The bacterium colony (a) and simple stain photo (b) of Fig. 1 bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701；
The solution starch transparent circle photo of Fig. 2 bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701；
The PCR amplification electrophoresis of the 16S rDNA of Fig. 3 bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701
Figure；
Bacillus amyloliquefaciens (B.amyloliquefaciens) the YB1701 system hair that Fig. 4 is constructed based on 16S rDNA
Educate tree；
Fig. 5 bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 optimal pH (a) and growth curve chart (b)；
Fig. 6 bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 is to rice seedling growth promotion photo；
Antibacterial photograph of Fig. 7 bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 to test plant pathogen
Piece (being successively respectively culture dish front and back sides photo in every figure): (a) Pyricularia oryzae (b) corn top-rotten disease bacterium (c) corn round spot
Germ (d) phytophthora capsici (e) botrytis cinerea (f) Pythium aphanidermatum (g) target leaf spot of sorghum bacterium (h) yellow fusarium germ
(i) stenocarpella maydis (g) Maize Curvularia (k) sunflower kernel cup fungi (l) Exserohilum turcicum (m) corn corruption mildew
Bacterium (n) Black bundle disease of maize bacterium (o) Apple Mould Core bacterium (p) Melon fusarium Wilt.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, it is not used to
Limit the present invention.
Experimental method used in following experiments is conventional method unless otherwise specified.
Material used in following experiments, reagent etc. commercially obtain unless otherwise specified.
Beef-protein medium: beef extract 3g, peptone 10g, NaCl 5g, agar powder 18-20g, water 1000mL,
PH 7.2-7.4,121 DEG C of sterilizing 20min.
LB culture medium: 7.0,121 DEG C of peptone 10g, NaCl 10g, yeast extract 5g, distilled water 1000mL, pH sterilizings
20min。
Minimal medium: MgSO4.7H2O 0.5g, CaCl20.02g, KH2PO41.0g, NH4NO31.0g, FeCl3
7.2,121 DEG C of sterilizing 20min of 0.05g, distilled water 1000mL, pH.
DF culture medium: KH2PO44g, Na2HPO46g, MgSO4·7H2O 0.2g, glucose 2g, sodium gluconate 2g, lemon
Lemon acid 2g, (NH4)2SO42g, component 1 and each 0.1mL of 2 solution of component (component 1:H3BO310mg, MnSO4·H2O
11.19mg CuSO4·5H2O 78.22mg, MoO310mg, ZnSO4·7H2O 124.6mg is dissolved in 100mL sterile purified water
In, -4 DEG C of preservations；Component 2:FeSO4·7H2O 100mg is dissolved in 10mL sterile purified water, -4 DEG C of preservations), distilled water
1000mL, pH 7.2.
ADF culture medium: (the NH in DF is replaced with 3mmol/LACC4)2SO4, it is only nitrogen source.
Starch basal medium: starch 2g, beef extract 3g, peptone 10g, NaCl 5g, agar powder 18-20g, distilled water
1000mL, pH 7.2-7.4,121 DEG C of sterilizing 20min.
PDA culture medium: potato 200g, sucrose (or glucose) 20g, agar 15-20g, distilled water 1000mL, pH are certainly
So, 121 DEG C of sterilizing 30min.
Hoagland nutrient solution: it takes 1.26g Hoagland nutrient solution (powder) that 0.845g calcium nitrate is added, is settled to
1000mL, 115 DEG C of sterilizing 20min.
Test plant pathogen: Black bundle disease of maize bacterium (Acremonium strictum)；Exserohilum turcicum
(Exserohilum turcicum)；Corn rotten mildew fungus (Pythium gramincola Subra)；D. carbonum
(Bipolaris zeicola)；Phytophthora capsici (Phytophthora capsici Leonian))；Botrytis cinerea
(Botrytis cinerea Pers.)；Apple Mould Core bacterium (Trichothecium roseum))；Pyricularia oryzae
(Pyricularia grisea)；Melon fusarium Wilt (Fusarium oxysporum f.sp.melonis)；Target leaf spot of sorghum
Bacterium (Bipolaris sorghicola)；Maize Curvularia (Curvularia lunata)；Stenocarpella maydis
(Fusarium graminearum)；Yellow fusarium germ (Fusarium culmorum)；Corn top-rotten disease bacterium (Frumentum
subglutinans)；Sunflower kernel cup fungi (Sclerotiniasclerotorium)；Pythium aphanidermatum (Pythium
aphanidermatum).Suppression of bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 to test plant pathogen
Bacterium photo is shown in Fig. 7.
The separation and identification of embodiment 1, bacillus amyloliquefaciens of the invention (B.amyloliquefaciens) YB1701
1. the separation of bacterial strain YB1701
It takes the rhizosphere of fresh acquisition to have the fluffy grass of Honghe fault alkali of soil, shakes off its foundation soil, weigh 10g, after air-drying
Pedotheque is placed in the conical flask with bead and equipped with 90mL sterile saline, room temperature 120rpmin oscillation
Soil supension is made in 30min.1mL soil supension is taken to carry out gradient dilution into the test tube that 9mL sterile saline is housed, point
Do not take 10-3, 10-4, 10-5Dilution 0.1mL is applied on screening and culturing medium plate, and for 24 hours, picking single colonie carries out for 37 DEG C of cultures
It isolates and purifies.
2. the identification of bacterial strain YB1701
(1) Morphological Identification
To in logarithmic phase and the stable bacillus amyloliquefaciens (B.amyloliquefaciens) of colonial morphology size
The single colonie of YB1701 carries out morphologic description, and whether size, color, transparency, bacterium colony configuration of surface including bacterium colony (put down
Smooth, fold etc.) and colony edge form (whether neatly, if rule etc.).
The bacillus amyloliquefaciens after simple stain, Gram's staining and spore staining are observed under an optical microscope
The thalli morphology of YB1701 (being in logarithmic growth phase) is under the jurisdiction of Gram-negative bacteria or positive bacteria, if tool gemma.
The result shows that milky is presented in bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 bacterium colony, it is impermeable
Bright, there is fold on surface, and edge is irregular, easy picking；Short and small rod-shaped, gram-positive bacteria is presented in thallus, and has gemma, thallus
A length of 1.6-3.1 μm, width is 0.9-1.1 μm (see Fig. 1 and Fig. 2).
(2) analysis of physio biochemical characteristics
With reference to " Berger bacterial identification manual " (Buchanan R.E., the lucky wood of N.E. this etc. write, Berger bacterial identification manual,
Institute of Microorganism, Academia Sinica's translation, Science Press, 1984) and " Microbiology Experiment " (Shen Ping, Fan Xiurong, Li Guang
The Beijing military Microbiology Experiment (third edition): Higher Education Publishing House, 1999) the above-mentioned bacillus amyloliquefaciens of measurement
(B.amyloliquefaciens) physiological and biochemical property of YB1701.
The physiological and biochemical property measurement result of the bacillus amyloliquefaciens YB1701 is as shown in table 1:
The physiological and biochemical property of 1 bacillus amyloliquefaciens of table (B.amyloliquefaciens) YB1701
Note: "+" indicates positive, and "-" indicates negative
3.16S rDNA sequence homology analysis
The template that bacterial strain total DNA is used as gene magnification is extracted using bacterium colony method, 16S rDNA universal primer is 27F:5'-
AGAGTTTGATCCTGGCTCAG-3'；1492r:5'-TACGGTTACCTTGTTACGACTT-3' carries out PCR amplification.Reactant
System are as follows: 2 × mixTaq (being purchased from TaKaRa company) 25uL, two kinds of each 1 μ L of primer (10 μM) expand 2 μ L of template, use ddH2O is mended
Sufficient system is to 50 μ L.Response procedures: 95 DEG C of initial denaturations need 1min, 95 DEG C of denaturation 1min after, 55 DEG C of annealing 1min, 72 DEG C of extensions
2min, then 35 recycle, and last 72 DEG C re-extend 10min, 4 DEG C of preservations.DNA sequencing is completed by the raw work in Shanghai, will be measured
Base sequence carry out homology analysis with EzBioCloud database.
The sequence of the 16S rDNA of bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 bacterial strain is detailed in sequence
Sequence in table.The agarose gel electrophoresis results and phylogenetic tree of the 16S rDNA of bacillus amyloliquefaciens YB1701 bacterial strain
See Fig. 3 and Fig. 4.
4. solving the measurement of starch ability
10uL is taken to be in the bacillus amyloliquefaciens YB1701 bacterium that the above-mentioned steps 1 of logarithmic growth phase isolate and purify out outstanding
Liquid, bikini are inoculated in the culture medium containing starch, and the tincture of iodine is added in plate after cultivating 2d, has seen whether after 10min
Transparent circle occurs.
The result shows that occurring apparent transparent circle on culture medium (see Fig. 2), it was demonstrated that the solution that above-mentioned steps 1 isolate and purify out
Bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 has very strong solution starch ability.
5. growth characteristics are analyzed
It uses beef extract-peptone fluid nutrient medium for basic culture medium, sets 4,5,6,7,8,9,10,11 for its pH,
3 repetitions of every group of setting.It is inoculated in culture medium according to 1% inoculum concentration, for 24 hours, sampling utilizes 37 DEG C of 180rpmin shaking table cultures
Nanodrop2000 micro-spectrophotometer carries out the measurement of OD value, with OD600For ordinate, different pH are abscissa, draw broken line
Figure such as Fig. 5 A.
The result shows that above-mentioned bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 bacterial strain isolated and purified
Optimal pH is pH7.0-8.0.
Embodiment 2, bacillus amyloliquefaciens of the invention (B.amyloliquefaciens) YB1701 growth curve draw
System
Using conventional beef extract-peptone bacteria culture media content as substrate, according to 1% inoculum concentration, in pH7.5, physiology salt
It spends under conditions of (NaCl) 1%, 37 DEG C of 180rpmin shaking table cultures, sampling interval is that 2h takes a sample, carries out OD600Measurement, often
3 groups of group setting is parallel.Sampling carries out the measurement of OD value using Nanodrop2000 micro-spectrophotometer, with OD600Numerical value is from seat
Mark, incubation time is abscissa, draws thalli growth curve such as Fig. 5 B.
The medium optimization of embodiment 3, bacillus amyloliquefaciens of the invention (B.amyloliquefaciens) YB1701
Use minimal medium for basic culture medium, respectively with 1% sucrose, maltose, lactose, glucose, solvable
Property starch and beef extract are as sole carbon source, and respectively with 1% peptone, tryptone, yeast extract, yeast extract, ox
Meat extract, ammonium sulfate, ammonium chloride and potassium nitrate determine optimum carbon source and nitrogen source as only nitrogen source.It is sieved according to single factor experiment
The concentration of the optimum carbon source and nitrogen source and potassium dihydrogen phosphate selected and the optimum range of pH are variance factor, using orthogonal examination
Carry out medium optimization is tested, determines the optimum proportioning of culture medium each component.
2 orthogonal test medium optimization of table
The result shows that maltose be most suitable carbon source, dosage 0.3%, yeast extract be most suitable nitrogen source, dosage 1.5%,
Beef extract is 1%, potassium dihydrogen phosphate 0.5%, Optimal pH 7.5.Influence bacillus amyloliquefaciens
(B.amyloliquefaciens) the factor size sequence of YB1701 strain growth are as follows: yeast extract > maltose > beef extract > pH
> potassium dihydrogen phosphate.
Embodiment 4, bacillus amyloliquefaciens of the invention (B.amyloliquefaciens) YB1701 produce IAA ability and
Deaminase activity determination
1. producing the measurement of IAA ability
Using 1 obtained bacillus amyloliquefaciens (B.amyloliquefaciens) of colorimetric method for determining the present embodiment
YB1701 bacterial strain produce IAA ability, by strain inoculated in DF culture medium overnight growth, be then transferred into the ammonia of color containing 0.1%L-
In the DF culture medium of acid.Bacterial strain cultivates 7d under the conditions of 28 DEG C, and then 8000rpm is centrifuged 10min.1mL suspension is mixed in
2mL Fe-H2SO4Solution (1mL0.5M FeCl3·6H2O is added to 75mL 6.13M H2SO4) in, dark places 45min.It surveys
Sample is measured in the absorbance of 450nm, the concentration of IAA is found out according to standard curve.
As the result is shown: the IAA of bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 described in embodiment 1
Activity is 137.58 μ g/mL.Therefore bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701, which has, produces
IAA ability.
2.ACC deaminase activity determination
1 obtained bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 of embodiment is accessed into 5mL beef
In cream peptone fluid nutrient medium, 28 DEG C of cultures are for 24 hours.Draw the beef extract-peptone fluid nutrient medium of 0.5mL bacterium solution to 60mL
In, equal conditions spread cultivation 36h, and 10min is centrifuged under the conditions of 4 DEG C of 8000rpm, abandon supernatant, collect thallus.It is not added with 15mL
(NH4)2SO4DF fluid nutrient medium wash twice, be resuspended in the ADF culture medium of the final concentration of 3mM of 24mL ACC, be placed in 28
DEG C culture for 24 hours, induction generate acc deaminase.After collecting thallus, with the Tris-HCl buffer by centrifugation of 20mL 0.1M pH 7.6
It washes twice, thallus is averagely assigned in three EP pipes, -20 DEG C of storages.Storage thallus is resuspended in 1mL 0.1M pH's 7.6
In Tris-HCl buffer, it is centrifuged 5min in 10000rpm, abandons supernatant, rapid oscillation, smudge cells.Take 100 μ L crude enzyme liquids 4
DEG C storage, for measuring protein concentration；Remaining crude enzyme liquid measures ACC deaminase activity immediately.Take 200 μ L crude enzyme liquids to 1.5mL
EP in, be added 33.4 μ L0.3M ACC mix, 30 DEG C of water-bath 15min, be added 1mL 0.56M HCl terminate reaction.Control:
(1) crude enzyme liquid is not added；(2) ACC is not added.10000rpm is centrifuged 5min, draws 1mL supernatant into test tube, 800 μ L are added
The 2,4-dinitrophenylhydrazine solution of the HCl of 0.56M and 300 μ L 0.2%, 30 DEG C of heat preservation 30min, the NaOH that 2mL 2M is added are molten
Liquid mixes.Supernatant is substituted with distilled water, other steps are identical, return to zero for spectrophotometer, measure the absorbance at 540nm
Value, the content of α -one butyric acid is calculated according to α -one butyric acid standard curve.Take appropriate crude enzyme liquid to be added in test tube, then plus PBS it is slow
Fliud flushing complements to 75 μ L, and 2mL Coomassie Brillant Blue solution is added, and after mixing concussion, develop the color 5min, measures the absorbance at 595nm
Value, the protein content of thallus is calculated according to bovine serum albumin(BSA) standard curve.Acc deaminase vigor (μm olmg-1·h-1)
=α -one butyric acid content (μm ol)/protein content (mg)/reaction time (h).Protein determination uses colorimetric method, pure with ox blood
Albumen is as substrate.Measurement result is 3 average values.
As the result is shown: the ACC of bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 described in embodiment 1
Deaminase active is 3.03 μm of olmg-1·h-1.Therefore the bacillus amyloliquefaciens (B.amyloliquefaciens)
YB1701 has ACC deaminase activity, and then plant drought, resistant to high temperatures, pest and disease damage invasion or heavy metal pollution can be improved etc.
Adverse circumstance ability.
Embodiment 5, bacillus amyloliquefaciens of the invention (B.amyloliquefaciens) YB1701 are true to pathogenic
The bacteriostasis rate of bacterium measures
Using 3 points of face-off methods to 1 obtained bacillus amyloliquefaciens (B.amyloliquefaciens) of embodiment
YB1701 carries out the measurement of antagonizing pathogenic fungi bacteriostasis rate, and concrete operations are as follows: respectively at 3 points (in positive triangle on PDA plate
Shape) inoculated plant disease fungus bacteria cake, middle position be inoculated with bacillus amyloliquefaciens (B.amyloliquefaciens
Strain G81) YB1701 fermentation liquid filter paper, every group of 3 repetition set in 28 DEG C of insulating box and cultivate 4-5d, measures each
The diameter for handling bacterium colony, finds out average value and calculates bacteriostasis rate (%).
Bacteriostasis rate %=(the pathogen heart at a distance from the heart (D)-[the strains tested heart at a distance from the pathogen heart (d)-for examination
The bacterial strain heart and pathogen vertical range (r)]) (D) * 100% at a distance from the/pathogen heart and the heart
The result shows that the fermentation liquid of bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 bacterial strain is to for examination 16
Black bundle disease of maize bacterium, Apple Mould Core bacterium, corn rotten mildew fungus, D. carbonum, capsicum epidemic disease in kind plant pathogenic fungi
Germ, tomato gray mould, Apple Mould Core bacterium, Pyricularia oryzae, Melon fusarium Wilt, sorghum target spot, Curvularia tikka, corncob
Maize ear rot bacterium, yellow fusarium, corn top-rotten disease bacterium, sunflower kernel cup fungi, Pythium aphanidermatum, bacteriostasis rate >=50%；Especially pair
Exserohilum turcicum and sunflower kernel cup fungi for examination, bacteriostasis rate >=80% (are shown in Table 3).
3 bacillus amyloliquefaciens of table (B.amyloliquefaciens) YB1701 fermentation liquid antagonizing pathogenic fungi bacteriostasis rate
Embodiment 6, bacillus amyloliquefaciens of the invention (B.amyloliquefaciens) YB1701 are to rice seedling
Growth promotion experiment
Size is close and the rice paddy seed of full seed for selection respectively, impregnates for 24 hours at 28 DEG C, then vernalization at 30 DEG C
For 24 hours, the seed of germination is seeded in equipped with Hogland nutrient solution and is covered on the plastic cup of 750mL of gauze, every group 3 times
It repeats.It is placed in 28 DEG C/22 DEG C of illumination box diurnal temperature, photoperiod 12h/12h, relative air humidity 80%, illumination
Intensity is 300 μM/(m2.s2) in culture.It is handled when seedling length to 2 one heart stage of leaf, the solution starch of 3% logarithmic phase is added
Bacillus (B.amyloliquefaciens) YB1701 fermentation liquid.Using add 3% beef-protein medium as pair
According to.10d and 16d measurement height of seedling, root long, seedling and root dry weight, the results are shown in Table 4 and Fig. 6 after processing.
Influence of table 4 bacillus amyloliquefaciens (B.amyloliquefaciens) the YB1701 fermentation liquid to rice seedling
The result shows that the rice children through bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 fermentation liquor treatment
Seedling growth is promoted, in addition to except root long and seedling dry weight, in 10d, difference is not significant compared with the control, remaining height of seedling, root long,
Seedling stem weight and fresh weight are dramatically increased in 10d and 16d.Illustrate the resulting bacillus amyloliquefaciens of the present embodiment 1
(B.amyloliquefaciens) YB1701 has significant ground facilitation to the growth of rice seedling.
Sequence table
<110>Shenyang Normal University
<120>a kind of Bacillus amyloliquefaciens strain and its application
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1012
<212> DNA
<213>bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<400> 1
cccctgcggg tcccctattc ggcggctggc tcctaaaggt tacctcaccg acttcgggtg 60
ttacaaactc tcgtggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg 120
gcatgctgat ccgcgattac tagcgattcc agcttcacgc agtcgagttg cagactgcga 180
tccgaactga gaacagattt gtgggattgg cttaacctcg cggtttcgct gccctttgtt 240
ctgtccattg tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc 300
cccaccttcc tccggtttgt caccggcagt caccttagag tgcccaactg aatgctggca 360
actaagatca agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 420
acgacaacca tgcaccacct gtcactctgc ccccgaaggg gacgtcctat ctctaggatt 480
gtcagaggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct 540
ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc agtcttgcga ccgtactccc 600
caggcggagt gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacactta 660
gcactcatcg tttacggcgt ggactaccag ggtatctaat cctgttcgct ccccacgctt 720
tcgctcctca gcgtcagtta cagaccagag agtcgccttc gccactggtg ttcctccaca 780
tctctaacgc atttcaccgc tacacgtgga aattccactc ttctctttct gcactcaagt 840
tccccagttt ccaatgaccc tccccggttg agccgggggc tttcacatca gacttaagaa 900
accgcctgcg agctctttac gcccaataat tcccgacaac gcttgcaccc ttacgtaatt 960
accgcgtctg ctggcacgta ttaaccgtgg cttctggtaa ggaccgttaa ag 1012
Claims (10)
1. a kind of Bacillus amyloliquefaciens strain, which is characterized in that the Bacillus amyloliquefaciens strain is YB1701, and described
Bacillus amyloliquefaciens strain YB1701 is preserved in Chinese microorganism strain preservation conservator's common micro-organisms center, and preservation is compiled
Number be CGMCC No.16003.
2. a kind of microbial inoculum, which is characterized in that the active constituent of the microbial inoculum is bacillus amyloliquefaciens described in claim 1
The fermentation liquid of YB1701 or bacillus amyloliquefaciens YB1701 or the metabolite of bacillus amyloliquefaciens YB1701.
3. microbial inoculum according to claim 2, which is characterized in that the microbial inoculum is following any microbial inoculums:
1) inhibit the microbial inoculum of plant pathogenic fungi；
2) microbial inoculum of plant pathogenic fungi disease is prevented and treated；
3) promote the microbial inoculum of plant growth；
4) microbial inoculum of stress resistance of plant is improved；
5) plant is reduced to the microbial inoculum of adverse circumstance sensibility；
6) microbial inoculum of 1-Aminocyclopropane-1-carboxylate deaminase is generated；
7) microbial inoculum of indole-3-acetic acid is generated；
The adverse circumstance is arid, high temperature, pest and disease damage invasion or heavy metal pollution.
4. microbial inoculum according to claim 3, which is characterized in that the plant pathogenic fungi is following at least one phytopathy
Fungal pathogens:
1) Black bundle disease of maize bacterium；
2) Exserohilum turcicum；
3) Pythium aphanidermatum；
4) corn rotten mildew fungus；
5) D. carbonum；
6) phytophthora capsici；
7) botrytis cinerea；
8) Apple Mould Core bacterium；
9) Pyricularia oryzae；
10) Melon fusarium Wilt；
11) target leaf spot of sorghum bacterium；
12) Maize Curvularia；
13) stenocarpella maydis；
14) yellow fusarium germ；
15) corn top-rotten disease bacterium；
16) sunflower kernel cup fungi.
5. a kind of biological organic fertilizer, which is characterized in that the active constituent of the biological organic fertilizer is Xie Dian described in claim 1
The metabolism of the fermentation liquid or bacillus amyloliquefaciens YB1701 of afnyloliquefaciens YB1701 or bacillus amyloliquefaciens YB1701 produces
Object.
6. a kind of cultural method of Bacillus amyloliquefaciens strain as described in claim 1, which is characterized in that including by Xie Dian
Afnyloliquefaciens YB1701 is for cultivating the step of cultivating in the culture medium of bacillus amyloliquefaciens.
7. the cultural method of Bacillus amyloliquefaciens strain as claimed in claim 6, which comprises the steps of:
1) it takes the rhizosphere of fresh acquisition to have the fluffy grass of alkali of soil, shakes off its foundation soil, and weigh 10g foundation soil；
2) after foundation soil air-dries, pedotheque is placed in bead and the conical flask of 90mL sterile saline is housed
In, room temperature 120rpmin vibrates 30min, and soil supension is made；
3) it takes 1mL soil supension to carry out gradient dilution into the test tube that 9mL sterile saline is housed, takes 10 respectively-3, 10-4,
10-5Dilution 0.1mL is applied on screening and culturing medium plate, and for 24 hours, picking single colonie is isolated and purified, and is obtained for 37 DEG C of cultures
Bacillus amyloliquefaciens YB1701.
8. the cultural method of Bacillus amyloliquefaciens strain according to claim 7, which is characterized in that the screening and culturing
Based component are as follows: beef extract 3g, peptone 10g, NaCl 5g, agar powder 18-20g, water 1000mL, pH 7.2-7.4,121 DEG C go out
Bacterium 20min.
9. microbial inoculum as claimed in claim 2, which is characterized in that the preparation of the fermentation liquid of the bacillus amyloliquefaciens YB1701
Include the following steps:
1) the bacillus amyloliquefaciens YB1701 cultivated will be purified by inclined plane inoculating into beef extract-peptone solid medium,
37 DEG C, cultivate for 24 hours under 180r/min；
2) bacterial strain by step 1) activation accesses in LB liquid medium, the shaken cultivation 48h in 37 DEG C, 180r/min shaking table,
Obtain seed liquor；
3) the resulting seed liquor of step 2) is inoculated in beef-protein medium according to 5% inoculum concentration, in 37 DEG C of 180r/
Shaken cultivation 48h in min shaking table obtains bacillus amyloliquefaciens YB1701 fermentation liquid.
10. microbial inoculum as claimed in claim 9, which is characterized in that solved in the fermentation liquid of the bacillus amyloliquefaciens YB1701
Bacillus amyloliquefaciens YB1701 thallus content is 1.0 × 106~1.0 × 108cfu/mL。
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