CN110438026A - A kind of bacillus amyloliquefaciens GLD-191 and its microbial inoculum, preparation method and application - Google Patents
A kind of bacillus amyloliquefaciens GLD-191 and its microbial inoculum, preparation method and application Download PDFInfo
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- CN110438026A CN110438026A CN201910473124.4A CN201910473124A CN110438026A CN 110438026 A CN110438026 A CN 110438026A CN 201910473124 A CN201910473124 A CN 201910473124A CN 110438026 A CN110438026 A CN 110438026A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The present invention relates to biological control and microbial-bacterial fertilizer fields, and in particular to a kind of bacillus amyloliquefaciens GLD-191 and its microbial inoculum, preparation method and application.The present invention provides a kind of bacillus amyloliquefaciens GLD-191, and bacillus amyloliquefaciens GLD-191 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 19th, 2018, and deposit number is CGMCC No.17011.The present invention also provides a kind of microbial bacterial agent and preparation method thereof of bacillus amyloliquefaciens GLD-191 preparation, the application of bacillus amyloliquefaciens GLD-191 and its microbial inoculum in control of plant disease.The present invention is by bacillus amyloliquefaciens GLD-191 and Radix Isatidis slag solid medium secondary fermentation, prepare new microbe agent, plant is improved to the utilization rate of nutriment in Radix Isatidis slag, bacillus amyloliquefaciens GLD-191 can produce the antibacterial material for having bacteriostatic activity to plant pathogenic fungi, beneficial microbe population can be increased, expand disease control spectrum.
Description
Technical field
The present invention relates to biological control and microbial-bacterial fertilizer fields, and in particular to a kind of bacillus amyloliquefaciens GLD-191
And its microbial inoculum, preparation method and application.
Background technique
Bacillus amyloliquefaciens itself have the ability for widely inhibiting fungi and bacterium, thus it is possible to vary flora ring around it
Border and type, thus become the microbe species of biological bactericide and microbial-bacterial fertilizer most potentiality to be exploited.It largely grinds in the world
Studying carefully discovery bacillus amyloliquefaciens can produce the antibacterial material for having bacteriostatic activity to plant pathogenic fungi, these antibacterial materials master
There is the antibiotic such as peptides, lipopeptid class, bacteriocin and high-molecular-weight protein class antibacterial material etc. of low molecular weight.
Radix Isatidis slag is byproduct of the antiviral agent after extracting, and is rich in organic matter abundant and nutriment, but
It is usually all abandoned as waste, is a kind of significant wastage of plant resources.In Radix Isatidis slag still containing a small amount of antiviral and
The activated product of disease resistance, and contain a large amount of plant organic matter and nutriment, if directly used, plant is still unable to get
Effective use, since Radix Isatidis slag contains the nutritional ingredients such as a large amount of organic substance and vegetable protein and fiber, can be used as giving birth to
Fungi-proofing culture medium raw material and absorption carrier.Therefore, if can be inoculated with and ferment by beneficial microbe, overcome disadvantages mentioned above, it can
Utilization rate of the raising plant to it.
Summary of the invention
It is an object of the invention to provide a kind of solution starch according to existing existing issue and the deficiencies in the prior art
Bacillus GLD-191 and its microbial inoculum, preparation method and application, it is anti-that bacillus amyloliquefaciens GLD-19 can be applied to plant disease
In controlling, bacillus amyloliquefaciens GLD-191 and Radix Isatidis slag solid medium secondary fermentation can be prepared with biological and ecological methods to prevent plant disease, pests, and erosion function
The new microbe agent of energy.The present invention to integrated control disease, improve soil organic matter content and microbial population with
And promote agricultural and sideline product to recycle and be of great significance, being especially in green agriculture development in agricultural production practice has extensively
General application prospect.
To achieve the above object, the present invention is achieved by following scheme:
The present invention provides a kind of bacillus amyloliquefaciens GLD-191, bacillus amyloliquefaciens GLD-191 bacterial strain preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.17011, preservation day
Phase is on December 19th, 2018, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The 16S rDNA nucleotide sequence of bacillus amyloliquefaciens GLD-191 is as shown in SEQ ID NO.1, systematic evolution tree
As shown in Figure 1, the bacterium is a kind of mesophilic, aerobic, sporiferous bacillus, physiological characteristic multiplicity, the bacterium is in nature
It is widely present, nontoxic to people and animals, free from environmental pollution, can generate a variety of antibiotic and enzyme (is because finding the strain earliest
Alpha-amylase can be secreted and named), there is broad spectrum antibiotic activity and extremely strong anti-adversity ability.
The present invention also provides a kind of microbial bacterial agents of bacillus amyloliquefaciens GLD-191 preparation.
Further, microbial bacterial agent is solid fungicide.
Further, solid fungicide is by bacillus amyloliquefaciens GLD-191 seed fermentation liquid and Radix Isatidis solid medium
It is made.
Further, microbial bacterial agent preparation method the following steps are included:
(1) bacillus amyloliquefaciens GLD-191 is inoculated into improvement nutrient broth fluid nutrient medium, is 200- with revolving speed
350rpm, 27-34 DEG C of shaking flask culture 48h, obtain seed liquor；
(2) seed liquor for obtaining step (1) accesses LB liquid medium by 3-10% volume ratio, carries out liquid fermentation life
It produces, fermentation condition are as follows: pH 6.5-7.8, fermentation temperature range are 27-34 DEG C, mixing speed 200-350rpm, ventilatory capacity
0.5:1, fermentation time 72-96h, work spore count >=1 × 10 of fermentation liquid9A/ml obtains bacillus amyloliquefaciens GLD-
191 seed fermentation liquid；
(3) bacillus amyloliquefaciens GLD-191 seed fermentation liquid seed fermentation liquid will be obtained by 60- obtained by step (2)
The amount of 100ml/Kg is inoculated into Radix Isatidis solid medium, carries out secondary solid fermentation maturity, turning 1 every other day in fermentation process
Secondary, fermentation temperature is 20-50 DEG C, is fermented 5-9 days, and after fermentation, the aeration-drying in the case where temperature is not higher than 70 DEG C makes water content
Control is 20% hereinafter, package encapsulation is spare to get solid fungicide.
Further, Radix Isatidis solid medium each component mass percent are as follows: Radix Isatidis slag 3-25%, dregs of beans 15-
40%, wheat bran 10-35%, yeast extract 15-30%, urea 0.3-1.5%, NaCl 1-5%, KCl 0.5-3%,
MgSO40.5-0.8%, CaCO31.2-1.9%, pH 6.5-8, Radix Isatidis slag are that Radix Isatidis diameter smashes it through 200-300 mesh
The granule fines obtained after sieve, medium matrix of the Radix Isatidis slag as bacillus amyloliquefaciens GLD-191, and serve as microbial inoculum
Absorption carrier.
Further, improvement nutrient broth fluid nutrient medium contains: beef extract 0.6%, peptone 0.7%, NaCl
0.3%, remaining is distilled water, pH 6.3-7.8；LB liquid medium contain peptone 10g, NaCl 5.0g, yeast extract 10g,
Distilled water 1000ml, pH 7.2.
Application of the bacillus amyloliquefaciens GLD-191 provided by the invention in control of plant disease.
Application of the microbial bacterial agent provided by the invention in control of plant disease.
Beneficial effects of the present invention:
Bacillus amyloliquefaciens GLD-191 is had broad spectrum antibiotic activity and extremely strong anti-adversity ability by the present invention, in plant
There is good application effect in disease control.The present invention is by bacillus amyloliquefaciens GLD-191 and Radix Isatidis slag solid culture
Base secondary fermentation prepares new microbe agent, and bacillus amyloliquefaciens GLD-191, which can produce, has suppression to plant pathogenic fungi
The active antibacterial material of bacterium.
Bacillus amyloliquefaciens GLD-191 and Radix Isatidis slag solid medium secondary fermentation can be prepared with biological and ecological methods to prevent plant disease, pests, and erosion
The new microbe agent of function.Radix Isatidis slag is byproduct of the antiviral agent after extracting, and is rich in organic matter abundant
Make the culture medium of biocontrol microorganisms using Radix Isatidis slag with nutriment and a small amount of antiviral and disease resistance activated product, the present invention
Raw material and absorption carrier improve plant to the utilization rate of nutriment in Radix Isatidis slag, solve Radix Isatidis slag and directly use
When plant can not utilize and it is ineffective, even will appear the problem of injury seedling, solve the problems, such as agricultural and sideline product waste utilization.
Bacillus amyloliquefaciens GLD-191 inoculation and fermentation, gained microbial bacterial agent can be used for preventing and treating west Radix Isatidis slag through the invention
Cucurbit wilt can also be applied in promoting plant growth and soil improvement.
The present invention is to integrated control disease, raising soil organic matter content and microbial population and promotes agricultural and sideline production
Product, which recycle, to be of great significance, and being especially in green agriculture development in agricultural production practice has wide application prospect.
Detailed description of the invention
The 16S rDNA sequential system that Fig. 1 is bacillus amyloliquefaciens GLD-191 of the present invention develops tree；
Fig. 2 is the plate dual test result of bacillus amyloliquefaciens GLD-191 of the present invention and withered germ of water-melon.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts all other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The separation of bacillus amyloliquefaciens GLD-191
Bacillus amyloliquefaciens GLD-191 is to separate to obtain from Part of The Qinling Mountains In Shaanxi Province virgin forest soil.
Specific method is: the virgin forest soil for being collected in Shaanxi Province, white clouds township, Taibai County is protected at room temperature through natural air drying
It deposits, the separation for Antagonistic Fungi.The 1g soil is diluted to 10-3,10-4,10-5 gradient with sterile water.It is each to draw 100 μ L extremely
LB culture medium is coated with uniform with glass spreader.After cultivating 48~72h in 28~30 DEG C of incubators, picking colony form is not
Same bacterium is isolated and purified.Using withered germ of water-melon as indicator bacteria, using tablet face-off method to the short of money of withered germ of water-melon
Antibacterial is screened, and after 28 DEG C of grown cultures case 6d, is selected the bacterial strain with inhibition zone and is carried out next step secondary screening.
The bacterial strain point after primary dcreening operation is connect on 4 angles at anomaly plate center 3.5cm using crossing method, in plate
Centre while moving into the agar block with withered germ of water-melon of diameter 4.5mm, after 28 DEG C of culture 6d, select with inhibition zone and
The lasting bacterial strain of antagonism.Measurement, the size for recording antibacterial circle diameter simultaneously calculate inhibiting rate.Inhibiting rate (%)=(control group
Pathogen colony diameter-processing group pathogen colony diameter)/control group pathogen colony diameter × 100%, as shown in Fig. 2.
Embodiment 2
The identification of bacillus amyloliquefaciens GLD-191
Physio-biochemical characteristics and 16SrDNA points are carried out by a conventional method to bacterial strain GLD-191 separated in embodiment 1
Analysis, is accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and be named as bacillus amyloliquefaciens
GLD-191。
Bacterial strain GLD-191 grows not chromogenesis on LB culture medium, bacterium colony is flat or round, milky is opaque,
Colony edge is irregular.There is fold on surface.Gram-positive, rod-shaped, formation gemma, gemma ellipse or cylindricality.Referring to " common thin
Dientification of bacteria handbook " carry out the Physiology and biochemistry identification of bacterial strain: mannitol test is positive, lactose test is positive, V-P test is positive,
7%NaCl growth test is negative, methyl red test is negative, indole test is positive, pH5.7 growth test is positive, citrate benefit
With test is positive, L-arabinose test is positive, Starch Hydrolysis test is positive, D- xylose test is positive, gelatin liquefaction test sun
Property, decompose that casein test is positive, lipase is negative, nitrate reduction test is positive, malonate utilizes negative, cellulose point
Solution is positive, the double hydrolysis of arginine are negative.
It will be sequenced after 16SrDNA amplified production recovery purifying, the 16S rDNA nucleosides of bacillus amyloliquefaciens GLD-191
Acid sequence as shown in SEQ ID NO.1, specifically:
SEQ ID NO.1:
<110>Shaanxi Lv Dun crop Science and Technology Ltd.
<120>a kind of bacillus amyloliquefaciens GLD-191 and its microbial inoculum, preparation method and application
<210>1
<211>1401
<212>DNA
<213>bacillus amyloliquefaciens GLD-191
AGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCG GCGGACGGGTGAGTAACACGTGGGTA
ACCTGCCTGTAAGACTGG GATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAAC CGCATGGTTCA
GACATAAAAGGTGGCTTCGGCTACCACTTACAGA TGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTC
ACC AAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACAC TGGGACTGAGACACGGCCCAGACTCCT
ACGGGAGGCAGCAGTA GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGC GTGAGTGATGAAGG
TTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTA
ACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA CGTAGGTGGCAAGCGTTGTCCGGAATTATTG
GGCGTAAAGGGCTC GCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACC GGGGAGGGTCATTGGA
AACTGGGGAACTTGAGTGCAGAAGAGG AGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGG AGG
AACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACG CTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATT
AGATACCCTG GTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTC CGCCCCTTAGTGCTGCAGC
TAACGCATTAAGCACTCCGCCTGGGG AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCC GCAC
AAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAG AACCTTACCAGGTCTTGACATCCTCTGACAATCCT
AGAGATAGGA CGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGT CAGCTCGTGTCGTGAGATGT
TGGGTTAAGTCCCGCAACGAGCGC AACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGT GACTG
CCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAA TCATCATGCCCCTTATGACCTGGGCTACACACGTGCT
ACAATGGA CAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCAC AAATCTGTTCTCAGTTCGGATCG
CAGTCTGCAACTCGACTGCGTG AAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAAT ACGTTCC
CGGGCCTTGTACACACCGCCCGTCACACCACGAGAGT TTGTAACACCCGAAGTCGGTGAGGTAACCTTT
Sequencing gained sequence carries out BLAST comparison by ncbi database, is carried out by MEGA5.2 software and mode bacterium
Phylogenetic Analysis.As shown in Figure 1, Antagonistic Fungi GLD-191 and bacillus amyloliquefaciens Bacillus
Amyloloquefaciens affiliation is closest, and bacterial strain GLD-191 is accredited as bacillus amyloliquefaciens (Bacillus
amyloliquefaciens).Bacillus amyloliquefaciens GLD-191 is protected on December 19th, 2018 in Chinese microorganism strain
Hiding administration committee's common micro-organisms center has carried out preservation, deposit number are as follows: CGMCC No. 17011.
Embodiment 3
Efficiency test of the bacillus amyloliquefaciens GLD-191 to watermelon blight
Using sterile water as negative control, carbendazim is compareed as common medicament, measures bacillus amyloliquefaciens GLD-191
To the control efficiency of potting watermelon blight.
Bacterial strain GLD-191 separated in embodiment 1 is put into sterilized containing physiological saline and has bead
Tool plug test tube in, then oscillator vibrates, and forms bacteria suspension, and the concentration of bacteria suspension is 1 × 109CFU/ml。
Respectively with sterile water, bacteria suspension, carbendazim (by operation instruction dilute 1000 times) to potting watermelon (totally 90 plants, often
30 plants of group) foliage-spray is carried out, 1 × 10 is inoculated with after 48h5The withered germ of water-melon spore suspension of a/mL.Dark moisturizing 72h,
25 DEG C of constant temperature incubations observe incidence.Control efficiency, control efficiency %=[(check plot disease incidence-place are calculated with disease incidence
Manage area's disease incidence)/check plot disease incidence] × 100.
Test result shows that sterile water control group preventive effect is 0 (morbidity strain number is 30), and comparison medicament carbendazim preventive effect is
63.3% (morbidity strain number is 11), bacillus amyloliquefaciens GLD-191 preventive effect are 86.7% (morbidity strain number is 4).Test result
Show that bacillus amyloliquefaciens GLD-191 has good preventive effect to watermelon blight, has to agricultural production practical application and refer to
Lead meaning.
Embodiment 4
With bacillus amyloliquefaciens GLD-191 prepare microbial bacterial agent, the specific following steps of preparation method:
(1) bacillus amyloliquefaciens GLD-191 is inoculated into improvement nutrient broth fluid nutrient medium, improves nutrient meat soup
Body culture medium contains: beef extract 0.6%, peptone 0.7%, NaCl 0.3%, remaining is distilled water, pH 6.3-7.8, to turn
Speed is 250rpm, 30 DEG C of shaking flask culture 48h, obtains seed liquor；
(2) seed liquor for obtaining step (1) accesses LB liquid medium by 5% volume ratio, and LB liquid medium contains
Have: peptone 10g, NaC l5.0g, yeast extract 10g, distilled water 1000 ml, pH 7.2 carry out liquid fermentation production, fermentation
Condition are as follows: pH6.5-7.8, fermentation temperature range are 30 DEG C, mixing speed 300rpm, ventilatory capacity 0.5:1, and fermentation time is
84h, work spore count >=1 × 10 of fermentation liquid9A/ml obtains bacillus amyloliquefaciens GLD-191 seed fermentation liquid；
(3) bacillus amyloliquefaciens GLD-191 seed fermentation liquid obtained by step (2) is inoculated into plate by the amount of 80ml/Kg
In blue root solid medium, secondary solid fermentation maturity, Radix Isatidis solid medium each component mass percent are carried out are as follows: plate is blue
Root slag 20%, dregs of beans 20%, wheat bran 30%, yeast extract 20%, urea 1%, NaCl 4%, KCl 3%, MgSO4
0.5%, CaCO31.5%, pH 6.5-8, turning every other day 1 time in fermentation process, fermentation temperature is 40 DEG C, is fermented 6 days, fermentation
After, the aeration-drying in the case where temperature is not higher than 70 DEG C makes water content control 20% hereinafter, package encapsulation is spare to get micro-
Bacteria agent.
Embodiment 5
Radix Isatidis slag microbial bacterial agent prevents and treats watermelon blight and promotes the field efficacy experiment of watermelon growing
In order to show microbial bacterial agent of the present invention, control efficiency to watermelon blight and growth effect is promoted to watermelon
Fruit indoors on the basis of pot experiment, has carried out field control effectiveness test.In Xibei Univ. of Agricultural & Forest Science & Technology experimental plot, setting two
Group processing: processing 1 is not administered as control；The microbial bacterial agent prepared in 2 application embodiment 4 of processing.Select the growth of watermelon seedling
Consistent melonry carries out soil treatment in conjunction with watermelon seedling base manure, 50Kg/ mus of usage amount, is observed continuously after processing 3 months, adjusts
Field incidence is looked into, log long according to 3 method of embodiment calculating field efficacy, and measurement watermelon strain.
Test result shows the disease index of Radix Isatidis slag microbial bacterial agent processing group significantly lower than blank control group, energy
Watermelon blight disease incidence is effectively reduced, field efficacy reaches 76.7%；Meanwhile Radix Isatidis microbial bacterial agent can promote watermelon
The growth of seedling, processing group is averaged, and strain is long to reach 0.98m, 0.11m higher than blank control group.
In the description of this specification, the description of reference term " one embodiment ", " example ", " specific example " etc. means
Specific features described in conjunction with this embodiment or example, structure, material live feature and are contained at least one implementation of the invention
In example or example.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example.
Moreover, particular features, structures, materials, or characteristics described can be in any one or more of the embodiments or examples to close
Suitable mode combines.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.There is no detailed for preferred embodiment
All details are described, also do not limit the specific embodiment that the invention is only.Obviously, according to the content of this specification, can make
Many modifications and variations.These embodiments are chosen and specifically described to this specification, is original in order to better explain the present invention
Reason and practical application, so that skilled artisan be enable to better understand and utilize the present invention.The present invention is only authorized
The limitation of sharp claim and its full scope and equivalent.
SEQ ID NO.1:
<110>Shaanxi Lv Dun crop Science and Technology Ltd.
<120>a kind of bacillus amyloliquefaciens GLD-191 and its microbial inoculum, preparation method and application
<210> 1
<211> 1401
<212> DNA
<213>bacillus amyloliquefaciens GLD-191
agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60
gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg 120
ttgtttgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac 180
ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga 240
cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300
gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360
aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc 420
ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480
atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540
ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600
tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660
ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 720
ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780
ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840
acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct 960
agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080
cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140
gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa 1200
gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag 1260
tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380
aagtcggtga ggtaaccttt 1400
Claims (10)
1. a kind of bacillus amyloliquefaciens GLD-191, which is characterized in that the bacillus amyloliquefaciens GLD-191 bacterial strain preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.17011, preservation day
Phase is on December 19th, 2018.
2. bacillus amyloliquefaciens GLD-191 according to claim 1, which is characterized in that bacillus amyloliquefaciens GLD-
191 16S rDNA nucleotide sequence is as shown in SEQ ID NO.1.
3. the microbial bacterial agent of bacillus amyloliquefaciens GLD-191 preparation according to claim 1.
4. microbial bacterial agent according to claim 3, which is characterized in that microbial bacterial agent is solid fungicide.
5. microbial bacterial agent according to claim 4, which is characterized in that the solid fungicide is by bacillus amyloliquefaciens
GLD-191 seed fermentation liquid and Radix Isatidis solid medium are made.
6. microbial bacterial agent according to claim 5, which is characterized in that preparation method includes the following steps:
(1) bacillus amyloliquefaciens GLD-191 is inoculated into improvement nutrient broth fluid nutrient medium, is 200- with revolving speed
350rpm, 27-34 DEG C of shaking flask culture 48h, obtain seed liquor；
(2) seed liquor for obtaining step (1) accesses LB liquid medium by 3-10% volume ratio, carries out liquid fermentation production,
Its fermentation condition are as follows: pH 6.5-7.8, fermentation temperature range are 27-34 DEG C, mixing speed 200-350rpm, ventilatory capacity 0.5:
1, fermentation time is 72-96 hours, work spore count >=1 × 10 of fermentation liquid9A/ml obtains bacillus amyloliquefaciens GLD-191
Seed fermentation liquid；
(3) bacillus amyloliquefaciens GLD-191 seed fermentation liquid obtained by step (2) is inoculated into plate by the amount of 60-100ml/Kg
In blue root solid medium, secondary solid fermentation maturity is carried out, turning every other day 1 time in fermentation process, fermentation temperature 20-50
DEG C, it ferments 5-9 days, after fermentation, the aeration-drying in the case where temperature is not higher than 70 DEG C makes water content control 20% hereinafter, packet
Dress sealing is spare to get solid fungicide.
7. microbial bacterial agent according to claim 6, which is characterized in that Radix Isatidis solid medium each component quality percentage
Than are as follows: Radix Isatidis slag 3-25%, dregs of beans 15-40%, wheat bran 10-35%, yeast extract 15-30%, urea 0.3-
1.5%, NaCl 1-5%, KCl 0.5-3%, MgSO40.5-0.8%, CaCO31.2-1.9%, pH 6.5-8, Radix Isatidis slag
The granule fines obtained after 200-300 mesh are smashed it through for Radix Isatidis diameter, Radix Isatidis slag is as bacillus amyloliquefaciens GLD-
191 medium matrix, and serve as the absorption carrier of microbial inoculum.
8. microbial bacterial agent according to claim 6, which is characterized in that improvement nutrient broth fluid nutrient medium contains: ox
Meat extract 0.6%, peptone 0.7%, NaCl 0.3%, remaining is distilled water, pH 6.3-7.8；LB liquid medium contains: egg
White peptone 10g, NaCl 5.0g, yeast extract 10g, distilled water 1000ml, pH 7.2.
9. application of the bacillus amyloliquefaciens GLD-191 according to claim 1 or 2 in control of plant disease.
10. application of the microbial bacterial agent in control of plant disease according to any one of claim 3-8.
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