CN106939290A - Bacillus subtilis HMB26553 and its application - Google Patents
Bacillus subtilis HMB26553 and its application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- 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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- 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
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- 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
Abstract
The invention discloses a kind of bacillus subtilis (Bacillus subtilis) bacterial strain HMB26553, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its deposit number is CGMCC No.13211.The invention also discloses the microbial bacterial agent produced using the bacterial strain, and their applications in the plant diseases such as preventing and treating cotton seedling blight.Bacillus subtilis HMB26553 of the present invention is high to the preventive effect of cotton seedling blight, and average preventive effect is more than 90.0%；Secondly, the lasting medicine of its microbial bacterial agent is good, and without problem of environmental pollution；In addition, HMB26553 antimicrobial spectrums are wide, except cotton rhizoctonia solani, also there is good inhibiting effect to verticillium dahliae, cotton-wilt fusarium, botrytis cinerea and cucumber target spot bacterium.
Description
Technical field
The invention belongs to field of biological control, and in particular to a kind of bacillus subtilis HMB26553, further relate to utilize and be somebody's turn to do
The microbial bacterial agent of bacillus subtilis production, and their applications in the diseases such as preventing and treating cotton seedling blight.
Background technology
Cotton seedling blight is a kind of cotton in seedling stage disease as caused by Rhizoctonia solani Kuhn (Rhizoctonia solani K ü hn)
Harmful, its cardinal symptom is：Infected before cotton seed sprouting and cause rotten kind, cotton seed infects before not being unearthed after sprouting and causes rotten bud；
Cotton seedling is aggrieved after being unearthed, and initial stage produces yellowish-brown scab in nearly native face base portion, and scab gradually extends the whole base portion of encirclement in obvious
Hang contracting, sick seedling lodging of wilting is withered；Pull up the cortex below sick seedling, basal part of stem to leave in soil, only deposit the rat-tail shape that point is threaded
Xylem；After cotyledon is aggrieved, yellowish-brown irregular shape scab is produced more in the middle part of cotyledon, often come off perforation.Cotton seedling blight is tight
Ghost image rings cotton and grown, and it is dead in flakes to often result in cotton seedling after morbidity, is urgent problem to be solved.
Cotton seedling blight is such as dressed seed using chemical agent, be coated or soaked seed, after planting based on chemical prevention prior to seeding
Sprayed with chemical agent, pouring root etc., but chemopreventive effects are poor, and also easily produced in the presence of pollution environment, long-term prescription
The problems such as raw resistance to the action of a drug and high cost.Biological control is because with specialization is strong, preventive effect is high, lasting medicine is good and is not present
The advantages of problem of environmental pollution, more and more paid attention in recent years.Currently used for the microorganism master of preventing and treating cotton seedling blight
Have：Trichoderma harzianum (CN104430549A), atrophy bacillus (CN104531545A), bacillus megaterium
(CN103320360A), bacillus amyloliquefaciens (CN105176893A；CN104498386A), bacillus subtilis
(CN105199997A；CN104770398A；CN104642390A；CN105340972A；), dinuclear bridged complex
(CN1952106A), Serratieae (CN103122330A), Pseudomonas chlororaphis (CN1932006A) and Pseudomonas fluorescens
(CN1058230A) etc..From the foregoing, it is mainly bacillus (Bacillus) for preventing and treating the microorganism of cotton seedling blight,
Reason is that bacillus has wide distribution, easily separated culture, can produce the stronger gemma of resistance, storage period length and user
Just the features such as；In addition, bacillus can produce endogenous spore, there is extremely strong anti-adversity ability, compared to other kinds of biocontrol microorganisms, more
Be conducive to the production and transportation of microbial inoculum, such as survive, colonize and breed in formulation environment.Therefore, bacillus is a kind of
Preferable Biocontrol microorganism.
Due to bio-diversity and biological common evolutionary characteristic, variety classes for pathogenic microorganism and constantly enter
Change is tackled, it is necessary to screen more different types of microorganisms, and screens the biological and ecological methods to prevent plant disease, pests, and erosion micro- life inhibited to pathogen
Thing is the most economical effective ways to pathogenic microorganism prevention.
The content of the invention
Present invention aims at a kind of bacillus subtilis strain is provided, the bacterial strain has efficient and wide sterilization spectrum etc. excellent
Point.
Another object of the present invention is to provide the microbial bacterial agent produced using above-mentioned bacillus subtilis.
3rd purpose of the invention is the preparation method for providing mentioned microorganism microbial inoculum.
4th purpose of the invention is to provide above-mentioned bacillus subtilis in the plant diseases such as preventing and treating cotton seedling blight
Purposes.
5th purpose of the invention is the use for providing mentioned microorganism microbial inoculum in the plant diseases such as preventing and treating cotton seedling blight
On the way.
The present invention is achieved through the following technical solutions：
A kind of bacillus subtilis (Bacillus subtilis) bacterial strain HMB26553, the bacterial strain is preserved in Chinese micro- life
Thing culture presevation administration committee common micro-organisms center, its deposit number is CGMCC No.13211.
Present invention also offers the microbial bacterial agent produced using above-mentioned bacillus subtilis HMB26553, its active component
For bacillus subtilis HMB26553 thalline；Also include its Extracellular metabolism.
Mentioned microorganism microbial inoculum can be liquid preparation or pulvis.
The preparation method of mentioned microorganism microbial inoculum, comprises the following steps：
(1) actication of culture：The HMB26553 bacterial strains of Cord blood are activated on LB plating mediums, picking single bacterium colony exists
On LB slant mediums, cultivated 10~16 hours at 25~35 DEG C, obtain the bacterial strain of activation；
(2) prepared by seed liquor：Inoculation that a ring step (1) activates is scraped to 100mL LB liquid with sterile oese
In body culture medium, cultivated 10~16 hours under conditions of 25~35 DEG C, shaking speed are 150~220rpm, obtain seed liquor；
(3) fermented and cultured：The seed liquor of step (2) is linked into corn flour Huang for 1%~3% ratio according to volume ratio
In soybean medium (pH value is 7.0), 25~35 DEG C, shaking speed be 150~220rpm under conditions of fermented and cultured 35~
40h, obtains zymotic fluid；
(4) thalline and Number of spores in detection zymotic fluid, treat that grown spore in zymotic fluid accounts for gemma and thalline sum
Stop fermented and cultured when 90%；Gained is HMB26553 liquid preparation.
The constituent of LB plating mediums or LB slant mediums described in above-mentioned preparation method step (1) and its again
Measuring ratio is：8~12g of tryptone, 4~6g of yeast extract, 4~6g of sodium chloride, 12~18g of agar powder, water 1000mL.
The constituent and its weight ratio of LB fluid nutrient mediums described in above-mentioned preparation method step (2) be：Tryptose
8~12g of peptone, 4~6g of yeast extract, 4~6g of sodium chloride, water 1000mL.
Described LB plating mediums, LB slant mediums and LB fluid nutrient mediums is conventionally prepared.
The constituent and its percentage by weight of corn flour soybean powder medium described in above-mentioned preparation method step (3)
For：Corn flour 1.0~3.0%, analysis for soybean powder 1.0~3.0%, NaCl 0.1~0.8%, MnSO4·H2O 0.5~1.0%, its
Yu Weishui.
The preparation method of described corn flour soybean powder medium, according to percentage by weight by corn flour, analysis for soybean powder, NaCl
And MnSO4·H2O is mixed, and is added water, and is adjusted pH, is stirred.
Mentioned microorganism microbial inoculum, its bacillus subtilis HMB26553 viable count is more than 17.0 × 108cfu/mL。
Above-mentioned bacillus subtilis HMB26553 is in preventing and treating cotton seedling blight, cotton verticillium wilt, cotton wilt, tomato ash
Application in mildew or Leaf Spot Caused by Corynespora cassiicola on Cucumber.
Mentioned microorganism microbial inoculum is in preventing and treating cotton seedling blight, cotton verticillium wilt, cotton wilt, graw mold of tomato or cucumber
Application in target spot.
The application method of mentioned microorganism microbial inoculum：Above-mentioned gained microbial bacterial agent is diluted with water into viable bacteria body number is
107Cfu/mL, in before sowing cotton seed by seed-soaking half an hour；Or adsorb above-mentioned gained microbial bacterial agent with calcium carbonate, make
Be made bacillus subtilis HMB26553 pulvis, in before sowing cotton seed according to pesticide-seeds ratio 1：10 seed dressings.
The screening separation process of HMB26553 bacterial strains
In July, 2012, Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's Plant Protection Institute was from Linzhi city of Tibet Autonomous Region Milin County south Yi Gou
Township woodland collection soil sample, takes back and 1g soil samples are weighed behind laboratory is put into 250mL sterilizing triangular flask, add sterilized water 100mL,
It is put on shaking table, 170rpm vibration 30min stand 2h, take supernatant 10mL to add in 50mL sterile centrifugation tubes, in 80 DEG C of constant temperature
Water-bath 30 minutes, then takes 1mL plus sterilized water 9mL, 10mL10-3Times edaphon suspension is then dilute by soil supension
It is interpreted into 10-4、10-5、10-6Times dilution, takes each μ L of concentration microorganism suspension 200 to be applied on LB culture medium flat plates, each concentration weight
It is multiple 3 times, in 30 DEG C of incubated 1d~3d, carry out the separation and purifying of bacterium.And using cotton seedling blight as target, pass through flat board
Face-off method, pot experiment method carry out the screening of biocontrol microorganisms.As a result therefrom filtering out one has good preventing and treating to cotton seedling blight
The bacterial strain of effect, is named as HMB26553.
The taxonomic identification of HMB26553 bacterial strains：
(1) identification by morphological characters
It is shaft-like that thalline is cultivated on LB culture mediums, and life in brood cell, brood cell is produced after culture 10h, and ellipse, cyst is not swollen
Greatly, acid-fast stain is negative, and no parasporal crystal can be moved, flagellum Zhousheng.On nutrient agar panel, the light breast of Initial stage of culture bacterium colony
White, purulence shape is circular, neat in edge, and bacterium colony protuberance is in steamed bun shape, surface wettability；Late stage of culture bacterium colony is faint yellow, and edge is not whole
Together, dry tack free has fold；Rule and cultivate on nutrient agar slopes, it is linear；Static gas wave refrigerator in liquid medium within, table
Face forms white mycoderm.These morphological features with《Common bacteria system identification handbook》(east show pearl etc. writes Science Presses
.2001 year) described in bacillus morphological feature it is basically identical, tentatively judge that HMB26553 bacterial strains belong to bacillus.
(2) classification of 16S rDNA Sequence Identifications is utilized
Using HMB26553 genomic DNA as template, performing PCR amplification is entered by primer of universal primer F27 and R1492, is obtained
Pcr amplification product；Wherein described primer sequence is：
27F：5 '-AGAGTTTGATCATGGCTCAG-3 ' (SEQ ID No.1),
R1492：5’-GGCTACCTTGTTACGACTT-3’(SEQ ID No.2).
16S rDNA PCR reaction systems (50 μ L) are:10×PCR Buffer(Mg2+)5μL；dNTP Mixture
(2.5mM)5μL；Taq (5U/ μ L) 1 μ L, 27F (10 μm of ol/L) 1 μ L, R1492 (10 μm of ol/L) 1 μ L；HMB26553 genomes
DNA 50ng；ddH2O complements to 50 μ L.PCR reaction condition is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1.5min,
30 circulations；72℃10min.Gained pcr amplification product is subjected to gel electrophoresis, Shanghai Sheng Gong bioengineering Co., Ltd is delivered
Sequencing, obtains HMB26553 16S rDNA sequences (see SEQ ID No.3).Gained HMB26553 16S rDNA sequences are existed
Carry out tetraploid rice in Genbank, the 16S rDNA homologys of results strain HMB26553 and bacillus reach 98%；
MEGA softwares (Molecular Evolutionary Genetics Analysis, Molecular Evolutionary Genetics analysis) structure is utilized simultaneously
Phylogenetic tree is built, together with as a result ((see Fig. 1)) HMB26553 is aggregated to bacillus, illustrates that HMB26553 belongs to gemma
Bacillus (Bacillus).
(3) the identification classification of gyrB gene orders is utilized
Using HMB26553 genomic DNAs template, bacillus gyrB gene degenerate primers gyrB-F and gyrB-R are utilized
Enter performing PCR amplification for primer, obtain pcr amplification product；The sequence of wherein described gyrB-F and gyrB-R primers is：
gyrB-F：5 '-TTGRCGGHRGYGGHTATAAAGT-3 ' (SEQ ID No.4),
gyrB-R：5’-TCCDCCSTCAGARTCWCCCTC-3’(SEQ ID No.5).
GyrB pcr amplification reaction system (50 μ L) is:10×PCR Buffer(Mg2+)5μL；dNTP Mixture
(2.5mM)5μL；Taq(5U/μL)1μL；GyrB-F (10 μm of ol/L) 1 μ L, gyrB-R (10 μm of ol/L) 1 μ L；HMB26553 genes
Group DNA 50ng；ddH2O complements to 50 μ L.PCR reaction condition is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min,
30 circulations；72℃10min.Amplified production is delivered into the sequencing of Shanghai Sheng Gong bioengineering Co., Ltd, HMB26553 bacterial strains are obtained
GyrB gene orders (see SEQ ID No.6).The gyrB gene orders of the HMB26553 bacterial strains of acquisition are entered in Genbank
Row tetraploid rice,.As a result the gyrB gene homology highests of HMB26553 and bacillus subtilis are found, are reached
97.5%；MEGA softwares (Molecular Evolutionary Genetics Analysis, Molecular Evolutionary Genetics are utilized simultaneously
Analysis) phylogenetic tree construction, together with as a result HMB26553 bacterial strains are aggregated to bacillus subtilis (see Fig. 2), illustrate this hair
Bright bacterial strain HMB26553 is bacillus subtilis (Bacillus subtilis), and is a new strains.
In summary morphological feature, the result of 16S rDNA and gyrB gene homology comparative analyses, it is known that
HMB26553 belongs to bacillus subtilis (Bacillus subtilis), and different with existing Bacillus strain, is one
Individual new bacillus subtilis strain.
The present invention has the advantage that and beneficial effect：(1) bacillus subtilis HMB26553 of the present invention is to cotton seedling blight
Prevention effect it is good, average preventive effect is more than 90.0%, and (2) bacillus subtilis HMB26553 of the present invention is to cotton rhizoctonia solani
Specialization is strong, and is not likely to produce the resistance to the action of a drug, and lasting medicine is good；(3) microbial bacterial agent of the present invention is to people, animal safety, without ring
Border pollution problem；(4) bacillus subtilis HMB26553 antimicrobial spectrums of the present invention are wide, except cotton rhizoctonia solani, also to cotton yellow
Wither germ, cotton-wilt fusarium, botrytis cinerea and cucumber target spot bacterium has good inhibition；(5) preparation side of the invention
Method is simple, low cost, using simple.
Biological deposits：Bacillus subtilis (Bacillus subtilis) bacterial strain HMB26553 of the present invention, oneself was in 2016
October 28 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is：Court of Beijing
Institute of Microorganism, Academia Sinica of the positive institute 3 of area's North Star West Road 1, deposit number is CGMCC No.13211.
Brief description of the drawings
Fig. 1 is the HMB26553 bacterial strain phylogenetic trees obtained according to 16S rDNA sequences.
Fig. 2 is the HMB26553 bacterial strain phylogenetic trees obtained according to gyrB gene orders.
Embodiment
The present invention is explained further with specific embodiment below, but does not constitute the limit to the present invention in any way
System.Experimental method in following embodiments, unless otherwise instructed, is conventional method；Percentage composition in following embodiments, such as
Without special instruction, weight percentage is.
The preparation of embodiment 1HMB26553 microbial bacterial agents
Carry out as follows：
(1) actication of culture：- 80 DEG C bacillus subtilis (Bacillus subtilis) bacterial strain will be stored in
HMB26553 (HMB26553 oneself the common micro- life of China Committee for Culture Collection of Microorganisms is preserved on October 28th, 2016
Thing center, deposit number is CGMCC No.13211) in LB plating mediums, (its constituent and its weight ratio are：Tryptose
Peptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL) on activated (30 DEG C), picking single bacterium colony exists
(its constituent and its weight ratio are LB slant mediums：Tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder
15g, water 1000mL) at 30 DEG C cultivate 12 hours, obtain the bacterial strain of activation；
(2) preparation of seed liquor：Making LB fluid nutrient mediums according to a conventional method, (its constituent and its weight ratio are：Pancreas
Peptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL), LB nutrient solution 100mL are loaded in 250mL triangular flasks, it is high
Press moist heat sterilization, the bacterial strain activated accessed in an oese step (1) after temperature drops to room temperature, in every bottle, 30 DEG C, shake
Shaken cultivation is carried out under conditions of bed rotating speed 180rpm 12 hours, obtains seed liquor；
(3) preparation of corn flour soybean powder medium：According to percentage by weight by corn flour 1.5%, analysis for soybean powder 2.0%,
NaCl 0.5%, MnSO4·H2O 0.6% is added to the water, and is uniformly mixed, and produces corn flour soybean powder medium；It is sub-packed in
In 500mL triangular flasks, every bottle of 200mL；Sterilizing 30 minutes is carried out to corn flour soybean powder medium at 121 DEG C, then cools to 30
DEG C, it is standby；
(4) fermented and cultured：Inoculation step (2) institute in the every bottle of corn flour soybean powder medium 200mL prepared to step (3)
The seed liquor 2mL obtained；Fermented and cultured 36 hours is carried out under the conditions of 30 DEG C, shaking speed 180rpm, later every 30 minutes from
Sampling carries out microscopy in triangular flask, and the brood cell in the visual field and total thalline number are counted, and calculates brood cell and lead that (brood cell leads (%)
=maturation brood cell number/(ripe brood cell's number+thalline number) × 100)；Brood cell, which leads, stops fermented and cultured when reaching 90%；Common fermentation is trained
Support 48 hours, obtain HMB26553 liquid preparation.
The HMB26553 bacterial strains of the present invention of embodiment 2 are tested the inhibitory action of cotton standing dead bacterium mycelial growth
(1) for examination cotton standing dead bacterium RH-2：Cotton standing dead bacterium RH-2 bacterial strains pick up from Handan in Hebei province Qiu Xian cotton standing dead
Diseased plant, isolates and purifies through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, Plant Protection College, Hebei Agricultural Univ. plant
Department of Pathology is accredited as Rhizoctonia solani Kuhn (Rhizoctonia solani), and Pathogenic Tests show as High pathogenicity.
(2) test method：
Flat board determination test：Cotton standing dead bacterium (Rhizoctonia solani) RH-2 is activated on PDA plate first
Culture 3 days, then in colony edge region, uses card punchBacterium piece is made in punching, and cotton standing dead bacterium bacterium piece is transferred
2.0 centimetres away from indicator bacteria bacterium piece are connected in another PDA plate center, then by the bacillus subtilis HMB26553 points after activation
Place, if blank control (not putting the cotton standing dead bacteria growing situation for connecing HMB26553 bacterial strains).It is incubated at 25 DEG C, treat blank pair
During according to whole culture dish will be covered with, control increment (colony radius) and the processing increment (inoculation of cotton standing dead bacterium are measured
Suppression growth radius after HMB26553), antagonism is represented with bacteriostasis rate.Its computing formula is：
Bacteriostasis rate (%)=(control increment-processing increment)/control increment × 100.
As a result (it is shown in Table 1)：Bacillus subtilis HMB26553 is to the inhibiting rate of cotton standing dead bacterium up to 69.2%；Illustrate withered grass
Bacillus HMB26553 has obvious inhibitory action to cotton standing dead bacterium mycelial growth, the life with preventing and treating cotton seedling blight
Anti- potentiality.
Antagonism test result of the HMB26553 bacterial strains of the present invention of table 1 to cotton standing dead bacterium
| Strain name | Compare increment (mm) | Handle increment (mm) | Bacteriostasis rate (%) |
| HMB26553 | 39.0 | 12.0 | 69.2 |
Preventive effect contrast test of the HMB26553 bacterial strains of the present invention of embodiment 3 to cotton seedling blight
(1) test process：
(1) microbial bacterial agent：HMB26553 liquid preparations prepared by embodiment 1 are diluted with water 50 times.
(2) blank control：Clear water
(2) test method：
By vermiculite and Cotton Soil according to 1：1 ratio is mixed, identical every other day then in 121 DEG C of high pressure moist heat sterilizations 1 hour
Condition sterilizes 1 time again.Rhizoctonia solani Kuhn RH-2 hyphal bodies suspension (about 10 is inoculated with 500 grams of soil7Individual/milliliter) 10 millis
Rise, in the plastic flowerpot that 20 centimetres of diameter is seated in after stirring, compacting.Cotton variety selects Jifeng 106, sows preceding real
Apply 50 times of water diluent seed soaking half an hour of HMB26553 liquid preparations of the preparation of example 1；Blank pair is used as using Seed soaking half an hour
According to.10 seeds are sowed after seed soaking per basin, sterile soil is covered, is normally cultivated in greenhouse.Investigated day by day simultaneously after emerging
Record processing and blank control damping-off morbidity strain number, when blank control damping-off is fully fallen ill, calculate the incidence of disease and preventing and treating
Effect.
As a result in (table 2) first batch results from pot experiment test, bacillus subtilis HMB26553 of the present invention is to cotton standing dead
The preventive effect of disease is 89.65%.In second lot results from pot experiment test, bacillus subtilis HMB26553 of the present invention is to cotton standing dead
The preventive effect of disease is 87.99%.Illustrate that bacillus subtilis HMB26553 of the present invention and its microbial bacterial agent have to cotton seedling blight
Good prevention effect.
The HMB26553 liquid preparations of the present invention of table 2 are to cotton seedling blight preventive effect comparative test result
Preventive effect contrast test of the HMB26553 bacterial strains of the present invention of embodiment 4 to cotton seedling blight
(1) test process：
(1) microbial bacterial agent：HMB26553 liquid preparations prepared by embodiment 1 are diluted with water 50 times.
(2) blank control：Clear water
(2) test method：
Prepare nursery soil：By vermiculite and Cotton Soil according to 1：1 ratio was mixed, in 121 DEG C of high pressure moist heat sterilizations 1 hour,
The same terms sterilize 1 time again every other day, and Temperature fall is standby.Cotton standing dead rhizoctonia Spawn incubation：Will be purchased in market complete full small
The grain of rice soaks 4 hours in clear water, drains moisture, is divided in 50 milliliters of centrifuge tubes, often 20 grams of pipe, and 121 DEG C of high pressures are damp and hot to go out
Bacterium half an hour；Often 4~5 pieces of pipe inoculation cotton standing dead rhizoctonia RH-2 mycelia block, mixing, is trained in 25 DEG C of constant incubators after cooling
Support 10 days, it is standby.The nursery soil for preparing is loaded in high 10 centimetres of 50 milliliters of centrifuge tubes to apart from the centimeters of ttom of pipe 2,
A cultured small rice grain for carrying cotton standing dead rhizoctonia RH-2 is placed in native face, continues to load nursery soil to 7 lis away from ttom of pipe
At rice, it is compacted, sowing.Cotton variety selects Jifeng 106,50 times of the HMB26553 liquid preparations prepared before sowing with embodiment 1
Water diluent is soaked seed half an hour；Blank control is used as using Seed soaking half an hour.After planting covering nursery soil, gently with hand pressure,
Centrifuge tube lid is covered, 25 DEG C is placed in, cultivates in the 12 hours dark temperature control control light thermostatic chambers of illumination in 12 hours, quantitative watering is adjusted
Save soil moisture.Remove centrifuge tube lid when seed is unearthed.Investigated day by day after emerging and record processing and blank control and stand withered
Disease morbidity strain number, when blank control damping-off is fully fallen ill, calculates the incidence of disease and prevention effect.
As a result the bacillus subtilis HMB26553 of the present invention that (is shown in Table 3) is 84.18% to the prevention effect of cotton seedling blight.
Illustrate that bacillus subtilis HMB26553 of the present invention and its microbial bacterial agent have good prevention effect to cotton seedling blight.
The HMB26553 bacterial strains of the present invention of table 3 are to cotton seedling blight preventive effect comparative test result
| Processing | Investigate strain number | Diseased plant number | The incidence of disease (%) | Preventive effect (%) |
| HMB26553 liquid preparations | 107 | 3 | 2.8b | 84.18 |
| Blank control | 96 | 17 | 17.7a | -- |
Field comparison test of the HMB26553 bacterial strains of the present invention of embodiment 5 to cotton seedling blight preventive effect
(1) test process：
(1) HMB26553 pulvis：According to 1：Carbon is added in the HMB26553 liquid preparations that 1 ratio is prepared to embodiment 1
Sour calcium, produces HMB26553 pulvis.
(2) blank control：Clear water
(2) test method：
This is tested is carried out in the village cotton test fields of Hebei province Baoding Gaoyang County Bei Yu eight.According to 1：1 ratio is to embodiment 1
The HMB26553 liquid preparations addition calcium carbonate of preparation, is made HMB26553 pulvis.Cotton variety selects Jifeng 106, before sowing
With HMB26553 pulvis according to pesticide-seeds ratio 1：10 seed dressings；Not handle seed as blank control.Each processing is repeated 4 times, with
Machine is arranged, 40 square metres of plot area.Observation unit's area number of emerging and damping-off dead seedling number after emerging, are investigated once for every 3 days,
The cumulative statistics cotton seedling blight incidence of disease, calculates prevention effect.
As a result (4 are shown in Table)：Bacillus subtilis HMB26553 of the present invention is 100.0% to the preventive effect of cotton seedling blight.Say
Bright bacillus subtilis HMB26553 of the present invention and its microbial bacterial agent have extraordinary prevention effect to cotton seedling blight.
The HMB26553 bacterial strains of the present invention of table 4 are to cotton seedling blight preventive effect comparative test result
| Processing | Investigate strain number | Diseased plant number | The incidence of disease (%) | Preventive effect (%) |
| HMB26553 | 174 | 0 | 0.0b | 100.0 |
| Blank control | 186 | 14 | 7.5a | -- |
The HMB26553 bacterial strains of the present invention of embodiment 6 are tested the inhibitory action of 4 kinds of plant pathogenic fungis
(1) for 4 kinds of disease funguses of examination and its source：
Verticillium Dahliae (Verticillium dahliae) VD-1, cotton-wilt fusarium (Fusarium oxysporium
F.sp.vasinfectum) FOV-1, botrytis cinerea (Botrytis cinerea) BC-32 and Leaf Spot Caused by Corynespora cassiicola on Cucumber bacterium
(Corynespora cassiicola)CC-1。
This 4 kinds of disease fungus bacterial strains are real from Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's biocontrol of plant disease
Test room.
(2) test method：
Detect that suppression of the bacillus subtilis HMB26553 bacterial strains to 4 kinds of plant pathogenic fungis is made using opposite culture method
With HMB26553 bacterial strains are activated into 24h at 30 DEG C on LB slant mediums, the HMB26553 bacterial strains of activation are obtained.By 4 kinds of cause of diseases
Fungi activates 5d in PDA culture medium, then plays the bacterium disk that takes cell age consistent, bacterium in colony edge diameter 6mm card punch
Silk is transferred to another PDA plate center down, HMB26553 bacterial strains is being inoculated with triangle disposition away from bacterium Pan2cmChu, not to be inoculated with
HMB26553 is control, often handles 3 repetitions, is put in 25 DEG C of incubated 5d, measurement processing colony radius and microbionation point
To the spacing (antibacterial band) of pathogen colony edge, bacteriostatic level is determined, bacteriostasis rate is calculated.
Bacteriostasis rate (%)=(control increment-processing increment)/control increment × 100.
Inhibitory action result of the test of the HMB26553 bacterial strains of the present invention of table 5 to 4 kinds of plant pathogenic fungis
As a result the HMB26553 bacterial strains of the present invention that (are shown in Table 5) to the bacteriostasis rate of 4 kinds of disease funguses for examination for 65.83%~
94.43%, wherein 84.43% has been reached to the bacteriostasis rate of verticillium dahliae, it is minimum to the bacteriostasis rate of cotton-wilt fusarium, be
65.83%, illustrate that bacterial strain HMB26553 antimicrobial spectrums of the present invention are wide.
SEQUENCE LISTING
<110>Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie
<120>Bacillus subtilis HMB26553 and its application
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 27F
<400> 1
agagtttgat catggctcag 20
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> R1492
<400> 2
ggctaccttg ttacgactt 19
<210> 3
<211> 969
<212> DNA
<213> Bacillus subtilis
<400> 3
cggagtggcg ggtgctatac atgcagtcga gcggacagat gggagcttgc tccctgatgt 60
tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc 180
ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaatggctc 240
accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420
gaacaagtgc cgttcgaata gggcggcacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540
cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600
agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660
cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840
cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960
gtcttgaca 969
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> gyrB-F
<400> 4
ttgrcgghrg ygghtataaa gt 22
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> gyrB-R
<400> 5
tccdccstca gartcwccct c 21
<210> 6
<211> 959
<212> DNA
<213> Bacillus subtilis
<400> 6
ctccttggat cggtgtagtg cgtcggtcgt aacgcactat caacagagct tgatgtgacg 60
gttcaccgtg acggtaaaat tcaccgccaa acctataaac gcggagttcc ggttacagac 120
cttgaaatca ttggcgaaac ggatcataca ggaacgacga cacattttgt cccggaccct 180
gaaattttct cagaaacaac cgagtatgat tacgatctgc ttgccaaccg cgtgcgtgaa 240
ttggcctttt taacaaaggg cgtaaacatc acgattgaag ataaacgtga aggacaagag 300
cgcaaaaatg aataccatta cgaaggcgga attaaaagtt atgtagagta tttaaaccgc 360
tctaaagagg ttgtccatga agagccgatt tacattgaag gcgaaaagga cggcattacg 420
gttgaagtgg ctttgcaata caatgacagc tacacaagca acatttactc gtttacaaac 480
aacattaaca cgtacgaagg cggtacccat gaagctggct tcaaaacggg cctgactcgt 540
gttatcaacg attacgccag aaaaaaaggg cttattaaag aaaatgatcc aaacctaagc 600
ggagatgacg taagggaagg gctgacagcg attatttcaa tcaaacaccc tgatccgcag 660
tttgagggcc aaacgaaaac aaagctgggc aactcagaag cacggacaat caccgatacg 720
ttattttcta cggcgatgga aacatttatg ctggaaaatc cagatgcggc caaaaaaatt 780
gtcgataaag gcttaatggc ggcaagagca agaatggctg cgaaaaaagc gcgtgaacta 840
acacgccgta agagtgcttt ggaaatttca aacttgcccg gtaagttagc ggactgctct 900
tcaaaagatc cgagcatttc cgagttatat atcgtagagg gagactcttg acggcggaa 959
Claims (9)
1. a kind of bacillus subtilis (Bacillus subtilis) bacterial strain HMB26553, the bacterial strain is preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, its deposit number is CGMCC No.13211.
2. utilize the microbial bacterial agent of the bacillus subtilis HMB26553 productions described in claim 1, it is characterised in that it is lived
Property composition be bacillus subtilis HMB26553 thalline.
3. microbial bacterial agent according to claim 2, it is characterised in that described microbial bacterial agent is liquid preparation or powder
Agent.
4. the preparation method of the microbial bacterial agent described in claim 2, it is characterised in that comprise the following steps：
(1) the HMB26553 bacterial strains of Cord blood are activated on LB plating mediums, picking single bacterium colony is in LB slant mediums
On, cultivated 10~16 hours at 25~35 DEG C, obtain the bacterial strain of activation；
(2) inoculation activated with sterile oese one ring step (1) of scraping is into 100mL LB fluid nutrient mediums, 25
~35 DEG C, shaking speed be 150~220rpm under conditions of cultivate 10~16 hours, obtain seed liquor；
(3) seed liquor of step (2) is linked into corn flour soybean powder medium (pH for 1%~3% ratio according to volume ratio
It is worth in 7.0), 35~40h of fermented and cultured under conditions of 25~35 DEG C, shaking speed are 150~220rpm obtains zymotic fluid；
(4) thalline and Number of spores in detection zymotic fluid, treat that grown spore accounts for the 90% of gemma and thalline sum in zymotic fluid
When stop fermented and cultured；Gained is HMB26553 liquid preparation.
5. preparation method according to claim 4, it is characterised in that LB plating mediums or LB described in its step (1)
Constituent and its weight ratio of slant medium is：8~12g of tryptone, 4~6g of yeast extract, 4~6g of sodium chloride,
12~18g of agar powder, water 1000mL.
6. preparation method according to claim 4, it is characterised in that the group of the LB fluid nutrient mediums described in its step (2)
It is into composition and its weight ratio：8~12g of tryptone, 4~6g of yeast extract, 4~6g of sodium chloride, water 1000mL.
7. preparation method according to claim 4, it is characterised in that the corn flour analysis for soybean powder culture described in its step (3)
The constituent and its percentage by weight of base be：Corn flour 1.0~3.0%, analysis for soybean powder 1.0~3.0%, NaCl 0.1~
0.8%, MnSO4·H2O 0.5~1.0%, remaining is water.
8. the bacillus subtilis HMB26553 described in claim 1 is withered in preventing and treating cotton seedling blight, cotton verticillium wilt, cotton
Application on disease, graw mold of tomato or Leaf Spot Caused by Corynespora cassiicola on Cucumber.
9. the microbial bacterial agent described in claim 2 is in preventing and treating cotton seedling blight, cotton verticillium wilt, cotton wilt, tomato ash
Application in mildew or Leaf Spot Caused by Corynespora cassiicola on Cucumber.
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| CN108841749A (en) * | 2018-06-28 | 2018-11-20 | 燕山大学 | The bacillus subtilis with wide antimicrobial spectrum of one plant of Salt-resistant alkali-resistant |
| CN108795829A (en) * | 2018-07-11 | 2018-11-13 | 金福赛（北京）生物科技有限公司 | Bacillus subtilis and its application in microbial inoculum and pork pig feed |
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| CN109486707B (en) * | 2018-11-23 | 2021-11-02 | 北京林业大学 | Bacillus subtilis strain and application thereof |
| CN112266889A (en) * | 2020-11-06 | 2021-01-26 | 青岛农业大学 | Pretreatment preparation for picking bacillus subtilis and edible fungi and application thereof |
| CN112266889B (en) * | 2020-11-06 | 2022-02-18 | 青岛农业大学 | Pretreatment preparation for picking bacillus subtilis and edible fungi and application thereof |
| CN114128725A (en) * | 2021-12-16 | 2022-03-04 | 四川省自然资源科学研究院 | Bacillus subtilis KT-10 and application thereof |
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