CN109355233A - A kind of bacillus amyloliquefaciens and its application - Google Patents
A kind of bacillus amyloliquefaciens and its application Download PDFInfo
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- CN109355233A CN109355233A CN201811473006.5A CN201811473006A CN109355233A CN 109355233 A CN109355233 A CN 109355233A CN 201811473006 A CN201811473006 A CN 201811473006A CN 109355233 A CN109355233 A CN 109355233A
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- 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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- 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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The present invention relates to biological control and technical field of bioengineering, specifically a kind of bacillus amyloliquefaciens SY-SF-01 and its application.Bacillus amyloliquefaciens are to be preserved in bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SY-SF-01 of China typical culture collection center, and deposit number is CCTCC M 2018264, and the preservation time is on May 14th, 2018.And it further provides the screening technique of bacillus amyloliquefaciens SY-SF-01 and its is inhibiting Rhizoctonia solani Kuhn, cucumber fusarium axysporum, Tobacco target spot bacterium, red bean anthrax bacteria, the application in verticillium dahliae.Bacterial strain of the present invention has very significant control efficiency to rice sheath blight disease, and living body preventive effect is up to 90% or more, also has good control efficiency to Tobacco target spot, in vitro bacteriostasis rate and living body potting preventive effect are up to 90% or more.
Description
Technical field
The present invention relates to biological control and technical field of bioengineering, specifically a kind of bacillus amyloliquefaciens SY-
SF-01 and its application.
Background technique
Rice sheath blight disease is one of the Major Diseases of the major rice region of one of rice disease important in the world and China,
Its pathogen be Rhizoctonia solani Kuhn (Rhizoctonia solani K ü hn), mainly with mycelium or sclerotium in the soil or
It is overwintering on sick straw and other host's residuums, there is the characteristics of strong septic and wide parasitics, pyrophilous high humidity.Rice sheath blight disease
It can all occur in rice entire breeding time, main harm leaf sheath and blade, fringe portion can be also endangered when serious and be deep into stalk.It should
Disease causes Seed-Setting Percentage in Rice to decline, and mass of 1000 kernel reduces, and the rice underproduction is up to 50%.
Tobacco target spot is the new expression reported at home for the first time by Wu Yuanhua etc., which is also by Rhizoctonia solani Kuhn
One of tobacco leaf position Major Diseases caused by (Rhizoctonia solani K ü hn), which occurs mainly in tobacco
Animated period and maturity period are a kind of short incubation periods, popular fast domestic tobacco new expression, the unexpected large area of the disease in 2005
Occur to plant cigarette district in Dandong City of Liaoning Province of China, be had brought tremendous economic losses to tobacco grower, then and in succession Northern Liaoning,
It is reported in succession in the area such as Guangxi and Heilungkiang.
Bacillus .spp. is a kind of aerobic or amphimicrobian, Gram-positive, sporiferous rod-shaped
Bacterium.The gemma that bacillus generates has the ability of the ambient pressure environments such as stronger resistance drying, thermal and uv radiation, simultaneously
Many of bacillus population has the bacterial strain of specific function, so in industry, agricultural, medicine and video processing and other fields
Have a wide range of applications.Bacillus amyloliquefaciens are one kinds of bacillus, are a kind of aerobic bacillus, have
Extensive antibacterial ability has a good application prospect in environmental protection, planting industry, culture fishery, degradable process hides solid waste
Object, degrading maize straws, alleviation broiler chicken liver oxidative damage, controlling plant diseases etc..
Currently, using bacillus controlling plant diseases at home and abroad it has been reported that the bacillus with bacteriostasis
Mainly have: bacillus subtilis (Bacillus.subtilis), bacillus amyloliquefaciens
(Bacillus.amyliliquefaciens), bacillus thuringiensis (Bacillus.thuringiensis), mostly viscous gemma
Bacillus (Bacillus.polymyxa), bacillus megaterium (Bacillus.megaterium) and bacillus pumilus
(Bacillus.pumilis) etc..Foreign countries are withered grass buds to studying in terms of the fungal diseases such as rice sheath blight disease biological controls more
Spore bacillus (Bacillus.subtilis), such as Bie etc. are separated and extracted from Bacillus.subtilis fermentation liquid
Fengycin analog can significantly inhibit cotton verticillium wilt；Lee.H etc. is from bacillus subtilis
(Bacillus.subtilis) a kind of lipopeptid bamylocin F is extracted in fermentation liquid, is had in terms of inhibiting the leaf blight of corn
Good effect.At home, Zheng Zhen separates and extracts antibacterial peptide from bacillus subtilis (Bacillus.subtilis) G-7
To prevent and treat banana blight；King wait quietly colonizing bacillus amyloliquefaciens (Bacillus.amyliliquefaciens) B6 kind
Eggplant root prevents and treats tomato wilt；The poplar winter waits quietly carrying out biology to sweet potato black rot using Bacillus amyloliquefaciens strain XZ-1
Prevention and treatment.It can be seen that bacillus controlling plant diseases are many kinds of, without foresight and corresponding enlightenment.
Summary of the invention
The purpose of the present invention is to provide a kind of bacillus amyloliquefaciens SY-SF-01 and its applications.
To achieve the above object, the invention adopts a technical scheme as:
A kind of bacillus amyloliquefaciens, it is characterised in that: bacillus amyloliquefaciens are to be preserved in Chinese Typical Representative culture guarantor
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SY-SF-01 at hiding center, deposit number are CCTCC M
2018264, the preservation time is on May 14th, 2018.
Bacterial strain SY-SF-01 is to isolate bacillus from the soil of Wuchang City of Heilongjiang Province paddy field；Bacterial strain warp
16S rDNA gene sequencing method is accredited as bacillus amyloliquefaciens；The morphological feature of bacillus amyloliquefaciens SY-SF-01: bacterial strain
SY-SF-01 is Gram positive aerobic bacterium, and the bacterium colony formed on peptone culture medium plate is rounded or subcircular, milky white
Color, it is glossy, extend with incubation time, bacterium colony has protrusion, and microscopically observation thallus is rod-shaped, endogenous spore.
A kind of bacillus amyloliquefaciens SY-SF-01 prevention and treatment rice sheath blight disease, cucumber fusarium axysporum, Tobacco target spot,
Application in red bean anthracnose or cotton verticillium wilt.
A kind of biocontrol agent, biocontrol agent contain the bacillus amyloliquefaciens SY-SF-01.
The bacillus amyloliquefaciens are the culture of the bacterial strain, culture concentrate, culture bacteria suspension, fermentation liquid or fermentation
Separate supernatant.
In the fermentation liquid, bacillus amyloliquefaciens viable count is not less than 1 × 108cfu/mL。
The fermentation liquid is to be inoculated in after bacterium activation in peptone fluid nutrient medium, in 120~200r/min,
At 25~30 DEG C shaken cultivation for 24 hours~72h is to get the fermentation liquid；
The culture concentrate is by above-mentioned fermentation liquid by centrifuge separation, cation exchange resin D101 absorption, 75%
Ethyl alcohol desorption, ethyl acetate extraction, can must cultivate concentrate.
The condition of culture for cultivating bacterial strain of the present invention is not particularly limited, and cultivation temperature, pH, hunting speed, liquid amount etc. are suitable
Close the strain growth.Typical temperature control in 25~30 DEG C, pH between 7.0-8.0, hunting speed 120~
200r/min, the general 250mL of liquid amount fill 80~120mL culture medium, cultivate 24~72 hours (preferably 48 hours), final thallus
Concentration is 1 × 108~1 × 1010cfu/mL。
The application of the biocontrol agent, the biocontrol agent is in prevention and treatment rice sheath blight disease, cucumber fusarium axysporum, tobacco target spot
Application in disease, red bean anthracnose or cotton verticillium wilt.
The application of the biocontrol agent, the biocontrol agent spray on rice or tobacco plant, for preventing and treating rice line
Blight or Tobacco target spot.
The present invention has the advantage that and the utility model has the advantages that
Bacillus amyloliquefaciens SY-SF-01 of the present invention has wider antimicrobial spectrum, and the crop of prevention and control is difficult to chemical agent
Soil-borne disease control efficiency is obvious, to Rhizoctonia solani Kuhn, cucumber fusarium axysporum, red bean anthrax bacteria, Tobacco target spot bacterium,
Cotton-wilt fusarium all has good inhibiting effect, and important value can be played in nuisanceless field crop and vegetable cultivation,
And low production cost, be readily produced, actual operation it is simple, application potential is big in terms of biocontrol of plant disease；
Specifically:
Bacillus amyloliquefaciens SY-SF-01 of the present invention is to Rhizoctonia solani Kuhn, cucumber fusarium axysporum, red bean anthracnose
A variety of germs such as bacterium, Tobacco target spot bacterium, cotton-wilt fusarium have apparent bacteriostasis, are up to the preventive effect of rice sheath blight disease
90% or more, the invention also discloses bacillus amyloliquefaciens SY-SF-01 to have significant control efficiency to rice sheath blight disease, living
Body biocontrol effect bacteriostasis rate is 93.2%, and, the indoor pot control efficiency and field control effect of rice sheath blight disease are reached
To 90% or more；In addition, bacteriostasis rate and living body potting control efficiency to Tobacco target spot, up to 90% or more, while to Huang
Cucurbit wilt also has good control efficiency.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, but protection scope of the present invention is not limited to
This.
Embodiment 1, the separation screening of bacterial strain SY-SF-01 and identification
Bacillus amyloliquefaciens SY-SF-01 is obtained by separating in the soil of Wuchang City of Heilongjiang Province paddy field.
Separation method: by the pedotheque 10g of acquisition in the 250mL triangular flask equipped with 100mL distilled water, it is put in shaking table
In vibrate 2d under the conditions of 28 DEG C, sample suspension is made, take 0.5mL sample suspension addition fill 4.5ml sterile water centrifugation
10 times are carried out in pipe to dilute step by step, respectively take 0.1mL 104、105、106、107Times dilution is coated on peptone culture medium (sucrose
30g, peptone 5g, FeSO4·7H2O 0.01g, MgSO4·7H2O 0.5g, KCl 0.5g, K2HPO41g, agar powder 20g steam
Distilled water 1000ml, pH=7.0~7.2) on plate, 2d is cultivated in 28 DEG C of constant incubators, according to form.Size, color etc.
Feature, 180 plants of picking single colonie, repeatedly scribing line purifying obtain pure lines bacterial strain altogether.
Screening technique: the bacteriostatic activity of in vitro plate opposite culture method measurement bacterium is taken, by Rhizoctonia solani Kuhn
It is inoculated in PDA culture medium plate central location, at bacteria cake or so 2.5cm, is carried out with the oese for being moistened with isolated strains
Scribing line culture, 28 DEG C of cultures measure the width of antibacterial band between pathogen and isolated strains afterwards for 24 hours, screen the bacterium of antibacterial bandwidth
Strain, obtained bacteriostasis is most strong, and the Strain Designation easily bred is SY-SF-01.
Identification method: bacterial strain SY-SF-01 is Gram positive aerobic bacterium, the bacterium formed on peptone culture medium plate
Rounded or subcircular is fallen, milky is glossy, extends with incubation time, and bacterium colony has protrusion, microscopically observation thallus bar
Shape, size are 0.6-0.8 μm of 1.0-1.2 μ m, endogenous spore.The 16S rDNA gene for expanding SY-SF-01 bacterial strain, measures it
16S rDNA gene order (see sequence table), logs inhttp://www.ncbi.nlm.nih.govWebsite, in GenBank into
Row nucleotide compares, and the affiliation of biocontrol microorganisms SY-SF-01 and Bacillusamyloliquefaciens is nearest as the result is shown.
Comprehensive analysis determines that bacterial strain SY-SF-01 is bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The 16S rDNA sequence of SY-SF-01 bacterial strain are as follows:
TGTCACTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTG
TTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGT
ATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGC
AGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGG
CTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGT
GTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCC
TCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCA
ACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG
ACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGG
GACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTC
TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCC
CGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGT
GCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTA
GCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCT
CCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC
GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAA
TTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCC
CCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGC
CCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGC
GGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGG
TGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTAC
GATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGT
CCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTG
TCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC
GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCAT
CTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAA
ACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACA
GGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGC
AAGCTCCCATCTGTCCGCTCGACTGCATGTATAGCACGCCC
The further plate fungistatic effect of embodiment 2, bacterial strain SY-SF-01
Fermentation medium: sucrose 30g, peptone 5g, FeSO4·7H2O 0.01g, MgSO4·7H2O0.5g, KCl
0.5g, K2HPO41g, distilled water 1000ml, pH=7.0-7.2
Biocontrol microorganisms SY-SF-01 fermentation liquid preparation: by the sterilized tooth of the pure bacterial strain of SY-SF-01 obtained in embodiment 1
Label are seeded in the 250ml triangular flask that liquid amount is 50ml, and at 28 DEG C, revolving speed is shaken cultivation on the constant-temperature table of 160r/min
48h obtains fermentation liquid, spare.
Pathogen Rhizoctonia solani Kuhn, cucumber fusarium axysporum, red bean anthrax bacteria, Tobacco target spot bacterium, cotton is withered
The germ that withers is inoculated in PDA culture medium, until covering with culture dish, gets bacteria cake from colony edge with the punch that diameter is 6mm, connects
Kind is in blank PDA culture medium plate central location, in the fermentation for being inoculated with 1 μ l biocontrol microorganisms SY-SF-01 away from the equidistant both sides of bacteria cake
Liquid is control with sterile water, and 27 DEG C of constant temperature incubations simultaneously observe opposite culture antibacterial situation, 3 repetitions of each processing, wait compare
When pathogen mycelia will cover with full plate in PDA culture medium plate, the growth radius of pathogen bacterium colony is measured, using following formula
Calculate the opposite bacteriostasis rate that bacterial strain SY-SF-01 grows pathogen fungi.
Opposite bacteriostasis rate=(control colony radius-processing colony radius)/control colony radius × 100%
Bacillus amyloliquefaciens SY-SF-01 is to Rhizoctonia solani Kuhn, cucumber fusarium axysporum, red bean anthrax bacteria, tobacco
Target spot germ, cotton-wilt fusarium have apparent bacteriostasis, the results are shown in Table 1, opposite bacteriostasis rate is respectively 93.2%,
87.5%, 89.5%, 92.9%, 89.7%.
Fungistatic effect of the 1 bacillus amyloliquefaciens SY-SF-01 of table to various pathogenic bacteria
The fermentation concentrated liquor preparation of embodiment 3, bacterial strain SY-SF-01
Bacillus amyloliquefaciens SY-SF-01 obtained in embodiment 1 is inoculated on peptone solid medium and activates bacterium
Strain, the bacterial strain after activation is seeded in the 250ml triangular flask that liquid amount is 50ml with sterilized toothpick, and at 28 DEG C, revolving speed is
For 24 hours, fermentation seed liquid is made in shaken cultivation on the constant-temperature table of 160r/min, and by seed liquor, 5% inoculum concentration is accessed by volume
Expanding fermentation medium (composition identical with fermentation medium in embodiment 2), temperature controls in 25~30 DEG C, revolving speed 120~
For 24 hours~72h is cultivated under the conditions of 200r/min and obtains fermentation liquid, and dilution-plate method detects cell concentration up to 1 × 108~1 × 1010cfu/
It is centrifuged under the conditions of mL, 1000r/min, takes centrifuged supernatant, adsorbed with nonpolar cation exchange resin D101, Mei Gesheng
There is addition 10g nonpolarity cation exchange resin D101 in the 1000ml triangular flask of 300ml centrifuged supernatant, oscillation for 24 hours, is filtered
Resin out, with 75% ethanol elution to colourless, eluent is extracted with ethyl acetate again, then molten with Rotary Evaporators removal extraction
Agent obtains concentrate, while being diluted fermentation concentrated liquor with sterile water, obtains fermentation 10 times of dilutions of concentrated liquor and fermentation liquid
100 times of dilutions of concentrate, it is spare.
Peptone solid medium: sucrose 30g, peptone 5g, FeSO4·7H2O 0.01g, MgSO4·7H2O 0.5g,
KCl 0.5g, K2HPO41g, agar powder 20g, distilled water 1000ml, pH=7.0~7.2
Fermentation medium: sucrose 30g, peptone 5g, FeSO4·7H2O 0.01g, MgSO4·7H2O0.5g, KCl
0.5g, K2HPO41g, distilled water 1000ml, pH=7.0~7.2
Embodiment 4, bacterial strain SY-SF-01 fermentation concentrated liquor are to the potting biocontrol effect of rice sheath blight disease
The Inoculated Rice on PDA solid medium (potato 200g, glucose 20g, agar powder 18g, 1L distilled water) plate
Sheath blight the original bacterium bacteria cake, 25 DEG C of constant temperature incubations are spare wait grow a large amount of sclerotium.
Rich 47 rice paddy seed of salt is seeded in pot for growing seedlings, it is long spare to the transplanting of one heart stage of 3 leaf to seedling.
By the fermentation concentrated liquor (stoste), fermentation 10 times of dilutions of concentrated liquor and hair of the 48h that ferments in above-described embodiment 3
100 times of dilutions of zymotic fluid concentrate are uniformly sprayed respectively after the consistent transplanting of growing way on the rice leaf of 20d, each processing 3
Secondary repetition, blank control group be spray equal amount of distilled water, for 24 hours after, rice banded sclerotial blight pathogen sclerotium is inoculated in leaf sheath, 10d
Spot expansion situation is investigated afterwards.
It is classified according to the damage symptom of rice leaf sheath and blade, rice sheath blight disease grade scale is as follows:
0 grade: complete stool is disease-free；
1 grade: the 4th blade and its following leaf sheath, blade morbidity (sword-like leave is the 1st leaf)；
3 grades: 3rd leaf and its following leaf sheath, blade morbidity；
5 grades: 2nd leaf and its following leaf sheath, blade morbidity；
7 grades: Flag Leaf Sheath and its following each leaf sheath, blade morbidity；
9 grades: complete stool morbidity, it is dead ahead of time.
Control efficiency of 2 bacterial strain SY-SF-01 of the table fermentation concentrated liquor to rice sheath blight disease
As shown in Table 2, the bacterial strain SY-SF-01 fermentation concentrated liquor that fermentation time is 48h has rice sheath blight disease good
Control efficiency.
Embodiment 5, fermentation time optimization
By embodiment 2 record fermentation concentrated liquor preparation method by fermentation time be set to for 24 hours, 36h, 48h, 60h,
72h, the fermentation concentrated liquor (stoste), fermentation 10 times of dilutions of concentrated liquor and fermentation liquid for obtaining the different fermentations time respectively are dense
100 times of dilutions of contracting liquid carry out the experiment in embodiment 3 again, and experimental result is as follows:
Control efficiency I of the bacterial strain SY-SF-01 fermentation concentrated liquor of 3 different fermentations time of table to rice sheath blight disease
Control efficiency II of the bacterial strain SY-SF-01 fermentation concentrated liquor of 4 different fermentations time of table to rice sheath blight disease
Control efficiency III of the bacterial strain SY-SF-01 fermentation concentrated liquor of 5 different fermentations time of table to rice sheath blight disease
Control efficiency IV of the bacterial strain SY-SF-01 fermentation concentrated liquor of 6 different fermentations time of table to rice sheath blight disease
Control efficiency V of the bacterial strain SY-SF-01 fermentation concentrated liquor of 7 different fermentations time of table to rice sheath blight disease
By 3~table of table 7 it is found that fermentation time for 24 hours~72h between, the fermentation concentrated liquor of bacterial strain SY-SF-01 is to water
The sheath and culm blight of rice has good control efficiency, is best with fermentation time 48h.
Embodiment 6, bacterial strain SY-SF-01 fermentation concentrated liquor are to the field biocontrol effect of rice sheath blight disease
Test is located at the defend the country paddy field in township of Wuchang City of Heilongjiang Province and carries out, this is experimental field many years Rice Cropping,
Rice varieties are Liaoxing No.1 (japonica rice), and rice sheath blight disease over the years has generation.Plot area is 320m2(16m × 20m),
Random district's groups arrangement, 3 repetitions of each processing, by the bacterial strain SY-SF-01 for the 48h that ferments in embodiment 3 ferment concentrated liquor and its
10 times and 100 times of dilutions are arranged by table 8, are administered using spray-on process, are attached most importance to Rice from middle and upper part and spray position, and 2018 8
The moon 1 was administered, and September in 2018 investigates the rice sheath blight disease incidence of each cell on the 1st, then calculates disease index and prevention and treatment is imitated
Fruit, disease index and control efficiency refer to " pesticide field efficacy medicine test criterion (one) bactericidal agent for preventing and treating rice sheath blight disease " GB/
T17980.20-2000 is counted and is calculated, and investigation result is as shown in table 9.
According to plant damage symptom grading, as unit of leaf, every random 5 points of samplings of cell, every investigation connected 4
Cave, totally 20 cave, records the total number of sheets, the sick number of sheets and sick series of investigation.
Rice sheath blight disease grade scale
0 grade: complete stool is disease-free；
1 grade: the morbidity of radical leaves leaf sheath；
2 grades: the following leaf sheath of third blade or blade morbidity (starting the first blade from top)；
The following leaf sheath of 3 grades: second blades or blade morbidity；
4 grades: top leaf sheath or top morbidity；
5 grades: complete stool morbidity, it is withered.
8 reagent agent experimental design of table
Field control effect of 9 bacterial strain SY-SF-01 of the table fermentation concentrated liquor to rice sheath blight disease
As shown in Table 9, bacillus amyloliquefaciens SY-SF-01 strain fermentation concentrated liquor also has field rice banded sclerotial blight
Good control efficiency.
Embodiment 7, bacterial strain SY-SF-01 fermentation concentrated liquor are to the potting biocontrol effect of Tobacco target spot
Tobacco is inoculated on PDA solid medium (potato 200g, glucose 20g, agar powder 18g, 1L distilled water) plate
Target spot pathogen bacteria cake, 26 DEG C of constant temperature incubations are to culture dish is covered with, for use.
When tobacco NC89 long is to 9 leaf phase, by the fermentation concentrated liquor stoste for the 48h that ferments in above-described embodiment 3, fermentation liquid
10 times of dilutions of concentrate and fermentation 100 times of dilutions of concentrated liquor are sprayed respectively on different tobacco plant blades, are sprayed
The clear water of equivalent is blank control, and 10 repetitions of each processing are inoculated with Tobacco target spot bacterium bacteria cake, bacterium using needle point method afterwards for 24 hours
Cake diameter is 6mm, every plant of inoculation the 5th and the 6th true leaf, 3 bacteria cakes of every inoculation, with the cotton moisturizing after sterilizing, after 48h
Bacteria cake and cotton, Investigate incidence and disease index after 7d are removed, and calculates preventive effect.
With reference to the method for the greenhouse artificial infection Tobacco target spot bacterium investigation lesion diameter of Zhao Yanqin etc., scab classification is divided
Standard are as follows: 0 grade: 0.0mm；1 grade: 0.0~1.0mm；3 grades of 1.1~2.0mm；5 grades: 2.0~5.0mm；7 grades: 2.01~9.0mm；
9 grades: > 9.01mm.
Control efficiency of 10 bacterial strain SY-SF-01 of the table fermentation concentrated liquor to Tobacco target spot
As shown in Table 8, fermentation time is the bacillus amyloliquefaciens SY-SF-01 strain fermentation concentrated liquor of 48h to tobacco
Target spot also has good control efficiency.
The above described specific embodiments of the present invention are not intended to limit the scope of the present invention..Any basis
Any other various changes and modifications made by technical concept of the invention should be included in the guarantor of the claims in the present invention
It protects in range.
Sequence table
<110>Shenyang Chemical Engineering Inst. Co., Ltd
<120>a kind of bacillus amyloliquefaciens and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1441
<212> DNA
<213>bacillus amyloliquefaciens SY-SF-01 (Bacillus amyloliquefaciens SY-SF-01)
<400> 1
tgtcacttcg gcggctggct ccataaaggt tacctcaccg acttcgggtg ttacaaactc 60
tcgtggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat 120
ccgcgattac tagcgattcc agcttcacgc agtcgagttg cagactgcga tccgaactga 180
gaacagattt gtgggattgg cttaacctcg cggtttcgct gccctttgtt ctgtccattg 240
tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc 300
tccggtttgt caccggcagt caccttagag tgcccaactg aatgctggca actaagatca 360
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca 420
tgcaccacct gtcactctgc ccccgaaggg gacgtcctat ctctaggatt gtcagaggat 480
gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg 540
tgcgggcccc cgtcaattcc tttgagtttc agtcttgcga ccgtactccc caggcggagt 600
gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacactta gcactcatcg 660
tttacggcgt ggactaccag ggtatctaat cctgttcgct ccccacgctt tcgctcctca 720
gcgtcagtta cagaccagag agtcgccttc gccactggtg ttcctccaca tctctacgca 780
tttcaccgct acacgtggaa ttccactctc ctcttctgca ctcaagttcc ccagtttcca 840
atgaccctcc ccggttgagc cgggggcttt cacatcagac ttaagaaacc gcctgcgagc 900
cctttacgcc caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 960
acgtagttag ccgtggcttt ctggttaggt accgtcaagg tgccgcccta tttgaacggc 1020
acttgttctt ccctaacaac agagctttac gatccgaaaa ccttcatcac tcacgcggcg 1080
ttgctccgtc agactttcgt ccattgcgga agattcccta ctgctgcctc ccgtaggagt 1140
ctgggccgtg tctcagtccc agtgtggccg atcaccctct caggtcggct acgcatcgtc 1200
gccttggtga gccgttacct caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg 1260
tagccgaagc caccttttat gtctgaacca tgcggttcaa acaaccatcc ggtattagcc 1320
ccggtttccc ggagttatcc cagtcttaca ggcaggttac ccacgtgtta ctcacccgtc 1380
cgccgctaac atcagggagc aagctcccat ctgtccgctc gactgcatgt atagcacgcc 1440
c 1441
Claims (8)
1. a kind of bacillus amyloliquefaciens, it is characterised in that: bacillus amyloliquefaciens are to be preserved in China typical culture collection
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SY-SF-01 at center, deposit number are CCTCC M
2018264, the preservation time is on May 14th, 2018.
2. bacillus amyloliquefaciens SY-SF-01 described in a kind of claim 1 is in prevention and treatment rice sheath blight disease, cucumber fusarium axysporum, tobacco
Application in target spot, red bean anthracnose or cotton verticillium wilt.
3. a kind of biocontrol agent, it is characterised in that: biocontrol agent is containing bacillus amyloliquefaciens SY-SF-01 described in claim 1.
4. biocontrol agent according to claim 3, which is characterized in that the bacillus amyloliquefaciens are the culture of the bacterial strain
Object, culture concentrate, culture bacteria suspension, fermentation liquid or fermentation separation supernatant.
5. biocontrol agent according to claim 4, which is characterized in that in the fermentation liquid, bacillus amyloliquefaciens viable count
Not less than 1 × 108cfu/mL。
6. biocontrol agent according to claim 4, which is characterized in that the fermentation liquid is to be inoculated in after bacterium activation
In peptone fluid nutrient medium, at 120~200r/min, 25~30 DEG C shaken cultivation for 24 hours~72h is to get the fermentation liquid；
The culture concentrate is by above-mentioned fermentation liquid by centrifuge separation, cation exchange resin D101 absorption, 75% ethyl alcohol
Desorption, ethyl acetate extraction, can must cultivate concentrate.
7. a kind of application of biocontrol agent described in claim 3, which is characterized in that the biocontrol agent is in prevention and treatment rice banded sclerotial blight
Application in disease, cucumber fusarium axysporum, Tobacco target spot, red bean anthracnose or cotton verticillium wilt.
8. by the application of biocontrol agent described in claim 7, which is characterized in that the biocontrol agent is sprayed in rice or tobacco plant
In strain, for preventing and treating rice sheath blight disease or Tobacco target spot.
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| CN115710144A (en) * | 2022-12-01 | 2023-02-24 | 贵州省烟草公司贵阳市公司 | Pesticide-fertilizer mixed combination for preventing and treating tobacco target spot disease and application thereof |
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