CN105199996B - A kind of bacillus amyloliquefaciens and its application for preventing graw mold of tomato - Google Patents
A kind of bacillus amyloliquefaciens and its application for preventing graw mold of tomato Download PDFInfo
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Abstract
The invention belongs to Strategies of Agricultural Bio-control fields, specifically disclose a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain HMB27633 for preventing graw mold of tomato, and deposit number is CGMCC No.11454.The invention also discloses using the bacterium prepare microbial bacterial agent and they prevention graw mold of tomato on purposes.Bacillus amyloliquefaciens HMB27633 biocidal properties of the present invention are strong, good to graw mold of tomato control effect, and durable antibacterial effect, stabilization；Secondly, microbial bacterial agent of the present invention belongs to environmentally friendly to people, animal safety；In addition, being not likely to produce drug resistance using the method for the present invention prevention graw mold of tomato；Bacillus amyloliquefaciens HMB27633 resistance also of the present invention is strong, and preparation method is simple, at low cost, easy to use.
Description
Technical field
The invention belongs to Strategies of Agricultural Bio-control fields, and in particular to a kind of solution starch gemma for preventing graw mold of tomato
Bacillus, and the microbial bacterial agent containing the bacillus amyloliquefaciens further relate to their purposes on prevention graw mold of tomato.
Background technology
Graw mold of tomato is a kind of important disease caused by the pathogen of Botrytis cinerea (Botrytis cinerea Pers.).It should
Disease main harm fruit, causes fruit rot, also endangers leaf, stem and flower, up to 20%~50%, loss is tight for the tomato underproduction
Weight.The pathogen host range is wider, in addition to infecting solanaceous vegetable, can also infect melon, wild cabbage, Kidney bean, lettuce, onion, apple
The crops such as fruit.
Currently, the main control method to graw mold of tomato has：First, chemical prevention.Though chemical prevention has quick, effect
The advantages that rate is high, but because be used for a long time can caused by environmental pollution, endanger the shortcomings of natural enemy of pest, therefore be gradually eliminated；
Followed by biological control, since biological control drug effect is high, the lasting period is long, is not likely to produce drug resistance, advantages of environment protection increasingly
It has been favored by people.
Bacillus (Bacillius sp) is a kind of biocontrol bacteria received significant attention, wide, easily separated with its distribution
Culture can generate the stronger gemma of resistance, the features such as storage period is long and easy to use, become a kind of micro- life of ideal biological and ecological methods to prevent plant disease, pests, and erosion
Object.Because bacillus can generate endogenous spore, there is extremely strong anti-adversity ability, compares other kinds of bio-control factors, be more advantageous to
The production of microbial inoculum is survived in formulation environment, colonizes and is bred.Therefore, the bud inhibited to pathogen is screened
Spore bacillus is most effectual way one of of the prevention in relation to plant disease.Bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) it is one of bacillus for preventing graw mold of tomato, is presently used for prevention graw mold of tomato
Bacillus amyloliquefaciens mainly have the CGMCC No.4777 (patent No.s：ZL201210059785.0), BA-KA3 (application numbers：
201410618581.5), SZ23 (application numbers：201410683826.2), LH-1 (number of patent application：201410735508.6) and
NCPSJ17 (application numbers：201310112580.9) etc..Since the diversity of pathogen is evolved with synchronous, it is therefore desirable to constantly seek
New bacterial strain is looked for prevent graw mold of tomato.
Invention content
Present invention aims at provide a kind of Bacillus amyloliquefaciens strain for preventing graw mold of tomato, bacterial strain tool
There is efficient, broad spectrum antibacterial activity advantage.
The present invention second is designed to provide the microbial bacterial agent using the production of above-mentioned bacillus amyloliquefaciens.
Third of the present invention is designed to provide the preparation method of mentioned microorganism microbial inoculum.
The present invention the 4th is designed to provide purposes of the above-mentioned bacillus amyloliquefaciens on prevention graw mold of tomato.
The present invention the 5th is designed to provide purposes of the mentioned microorganism microbial inoculum on prevention graw mold of tomato.
The present invention the 6th is designed to provide the identification method of above-mentioned bacillus amyloliquefaciens.
The present invention the 7th is designed to provide the identification method of mentioned microorganism microbial inoculum.
The invention is realized by the following technical scheme：
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain HMB27633, oneself was in 2015
September is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 29th, and (preservation address is：Beijing's southern exposure
No. 3 Institute of Microorganism, Academia Sinica of institute of area North Star West Road 1), deposit number is CGMCC No.11454.
The microbial bacterial agent produced using above-mentioned bacillus amyloliquefaciens, active constituent are HMB27633 thalline.
Mentioned microorganism microbial inoculum can be liquid preparation or pulvis.
The viable count of HMB27633 is more than 21.0 × 10 in mentioned microorganism microbial inoculum8cfu/mL。
The preparation method of mentioned microorganism microbial inoculum, includes the following steps：
(1) actication of culture：The HMB27633 bacterial strains of Cord blood are activated on LB plating mediums；Picking single bacterium colony exists
On LB slant mediums, is cultivated 10~16 hours at 25~35 DEG C, obtain the bacterial strain of activation；
(2) prepared by seed liquor：The inoculation of a ring step (1) activation is scraped to 100mL LB liquid with sterile oese
In body culture medium, in 25~35 DEG C, rotating speed to be cultivated 10~16 hours under the conditions of 150~220rpm, seed liquor is obtained；
(3) fermented and cultured：Seed liquor obtained by step (2) is linked into corn flour by the ratio for being 1~3% according to volume ratio
In soybean powder medium, 40~50h of fermented and cultured under the conditions of temperature is 25~35 DEG C, rotating speed is 150~220rpm must ferment
Liquid；
(4) thalline and Number of spores in zymotic fluid are detected, waits for that grown spore in zymotic fluid accounts for gemma and thalline sum
Stop fermented and cultured when 90%, gained is the liquid preparation of HMB27633 bacterial strains.
LB plating mediums, LB slant mediums and LB liquid medium described in above-mentioned preparation method is according to routine
It is prepared by method.
LB plating mediums described in above-mentioned preparation method step (1), constituent and its weight ratio are：Tryptose
8~12g of peptone, 4~6g of yeast extract, 4~6g of sodium chloride, 12~18g of agar powder, water 1000mL.
LB slant mediums described in above-mentioned preparation method step (1), constituent and its weight ratio are：Tryptose
8~12g of peptone, 4~6g of yeast extract, 4~6g of sodium chloride, 12~18g of agar powder, water 1000mL.
LB liquid medium described in above-mentioned preparation method step (2), constituent and its weight ratio are：Tryptose
8~12g of peptone, 4~6g of yeast extract, 4~6g of sodium chloride, water 1000mL.
Corn flour soybean powder medium described in above-mentioned preparation method step (3), constituent and its weight percent
Than for：Corn flour 1.0~3.0%, analysis for soybean powder 1.0~3.0%, NaCl 0.1~1.0%, MnSO4·H2O 0.5~1.0%,
Remaining is water；PH value is 7.2.
The preparation method of the corn flour soybean powder medium, including according to weight percent by corn flour, analysis for soybean powder,
NaCl and MnSO4·H2O is mixed, and is added water, and is adjusted pH and is stirred evenly.
Applications of the above-mentioned bacillus amyloliquefaciens HMB27633 on prevention graw mold of tomato.
Application of the mentioned microorganism microbial inoculum on prevention graw mold of tomato.
The application method of mentioned microorganism microbial inoculum：Above-mentioned gained microbial bacterial agent, which is diluted with water to viable bacteria body number, is
107Cfu/mL carries out foliar spray before the onset of graw mold of tomato, you can reaches and prevents the pathogenetic purpose of tomato gray mould.
The screening separation process of HMB27633 bacterial strains
HMB27633 bacterial strains are Cotton Soil of the Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie from Jingzhou City of Hubei Province
In it is isolated.In April, 2013, Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's Plant Protection Institute was adopted for 5 points from Jingzhou City of Hubei Province cotton field
Collect soil sample, weighing 1g after mixing is put into the sterilizing triangular flask of 250mL, and 100mL sterile waters are added, are put on shaking table, 170r/
Min vibrates 30min, stands 2h, and supernatant 10mL is taken to be added in 50mL sterile centrifugation tubes, in 80 DEG C of waters bath with thermostatic control 30 minutes, so
After take 1mL to add sterile water 9mL, 10mL10-3Times edaphon suspension, is then diluted to 10 by soil supension-4、10-5、
10-6Times dilution, takes each 200 μ L of concentration microorganism suspension to be applied on LB culture medium flat plates, each concentration is repeated 3 times, at 30 DEG C
Constant temperature incubation 1d~3d carries out the separation and purifying of bacterium.And using graw mold of tomato as target, by tablet face-off method, in vitro
Blade method, pot experiment method carry out the screening of biocontrol microorganisms.As a result therefrom filter out one has apparent prevention to graw mold of tomato
The bacterial strain of effect is named as HMB27633.
The taxonomic identification of HMB27633 bacterial strains：
(1) identification by morphological characters
It is rod-shaped that thalline is cultivated on LB culture mediums, and brood cell is generated after cultivating 10h, raw in brood cell, ellipse, and cyst is not swollen
Greatly, acid-fast stain is negative, and no parasporal crystal can move, flagellum Zhousheng.On nutrient agar panel, the light breast of Initial stage of culture bacterium colony
White, purulence shape is round, 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；It crosses and cultivates on nutrient agar slopes, it is linear；Static gas wave refrigerator in liquid medium, 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 almost the same, tentatively judge that bacterial strain HMB27633 belongs to bacillus.
(2) 16S rDNA Sequence Identifications are utilized to classify
Using the genomic DNA of HMB27633 as template, PCR amplification is carried out by primer pair 16S rDNA of F27 and R1492,
The primer sequence is：
F27：5’AGAGTTTGATCATGGCTCAG3’；(SEQ ID No:1)
R1492：5’GGCTACCTTGTTACGACTT3’；(SEQ ID No:2)
The amplification reaction system of 16S rDNA is 50 μ L:10×PCR Buffer(Mg2+)5μL；dNTP Mixture
(2.5mM)5μL；Taq (5U/ μ L) 1 μ L, F27 (10 μm of ol/L) 1 μ L, R1492 (10 μm of ol/L) 1 μ L；The genome of HMB27633
DNA 50ng；ddH2O complements to 50 μ L.The reaction condition of PCR is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1.5min,
30 cycles；72℃10min.Gained pcr amplification product is subjected to gel electrophoresis, delivers Shanghai Sheng Gong bioengineering Co., Ltd
Sequencing, obtains the 16S rDNA sequences of HMB27633 (see SEQ ID No:3).The 16S rDNA sequences of gained HMB27633 are existed
Carry out tetraploid rice in Genbank, the 16S rDNA homologys of results strain HMB27633 and bacillus reach 99%；
Phylogenetic tree construction (see Fig. 1), HMB27633 are aggregated to together with bacillus, illustrate that HMB27633 belongs to bacillus
Belong to (Bacillus).
(3) the identification classification of gyrB gene orders is utilized
Using HMB27633 genomic DNAs template, bacillus gyrB gene degenerate primers gyrB-F and gyrB-R are utilized
PCR amplification is carried out for primer, obtains pcr amplification product；The sequence of the wherein described gyrB-F and gyrB-R primers is：
gyrB-F：5’TTGRCGGHRGYGGHTATAAAGT3’；(SEQ ID No:4)
gyrB-R：5’TCCDCCSTCAGARTCWCCCTC3’；(SEQ ID No:5)
The pcr amplification reaction system of gyrB is 50 μ L: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；HMB27633 genes
Group DNA 50ng；ddH2O complements to 50 μ L.The reaction condition of PCR is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min,
30 cycles；72℃10min.Amplified production is delivered into the sequencing of Shanghai Sheng Gong bioengineering Co., Ltd, obtains HMB27633 bacterial strains
GyrB gene orders (see SEQ ID No:6).By the gyrB gene orders of the HMB27633 bacterial strains of acquisition in Genbank into
Row tetraploid rice, at the same using MEGA softwares (Molecular Evolutionary Genetics Analysis, molecule into
Change genetic analysis) phylogenetic tree construction.As a result, it has been found that the gyrB gene orders of HMB27633 and bacillus amyloliquefaciens are homologous
Property highest, reaches 99.29%；Phylogenetic tree construction (see Fig. 2), HMB27633 bacterial strains are aggregated to one with bacillus amyloliquefaciens
It rises, illustrates that HMB27633 is bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and be a new strains.
In summary morphological feature, 16S rDNA and gyrB gene homology comparative analyses as a result, understanding
HMB27633 belongs to bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and with existing solution starch gemma
Bacillus strain is different, is a new strains.
The identification method of above-mentioned Bacillus amyloliquefaciens strain HMB27633, including using strain to be tested genomic DNA as mould
Plate carries out PCR amplification by primer of F27 and R1492, if gained pcr amplification product is SEQ ID No:Nucleotide shown in 3
Sequence, as HMB27633 bacterial strains.
The identification method of above-mentioned Bacillus amyloliquefaciens strain HMB27633, including using strain to be tested genomic DNA as mould
Plate carries out PCR amplification by primer of gyrB-F and gyrB-R, if gained pcr amplification product is SEQ ID No:Core shown in 6
Nucleotide sequence, as HMB27633 bacterial strains.
The identification method of mentioned microorganism microbial inoculum, it is identical as the identification method of HMB27633 bacterial strains, i.e., in microbial inoculum to be measured
The genomic DNA of extraction is template, and PCR amplification is carried out by primer of F27 and R1492, if gained pcr amplification product is SEQ
ID No:Nucleotide sequence shown in 3, as HMB27633 bacterial strains；Or the genomic DNA to be extracted in microbial inoculum to be measured is template,
PCR amplification is carried out by primer of gyrB-F and gyrB-R, if gained pcr amplification product is SEQ ID No:Nucleosides shown in 6
Acid sequence, as HMB27633 bacterial strains.
The present invention has the advantage that and advantageous effect：(1) bacillus amyloliquefaciens HMB27633 biocidal properties of the present invention are strong, right
Graw mold of tomato control effect is good, and average preventive effect is 80.0% or more, and durable antibacterial effect, stabilization；(2) microorganism of the present invention
Microbial inoculum belongs to environmentally friendly to people, animal safety；(3) it is not likely to produce drug resistance using the method for the present invention prevention graw mold of tomato；
(4) bacillus amyloliquefaciens HMB27633 resistance of the present invention is strong, and preparation method is simple, at low cost, easy to use.
Description of the drawings
Fig. 1 are the systematic growth tree graph that HMB27633 bacterial strains are obtained according to 16S rDNA sequences.
Fig. 2 are the systematic growth tree graph that HMB27633 bacterial strains are obtained according to gyrB gene orders.
Specific implementation mode
The present invention is further clearly explained with specific embodiment below, but is not constituted in any way to the present invention
Limitation.Experimental method in following embodiments is unless otherwise instructed conventional method；Percentage in following embodiments contains
Amount, is weight percentage unless otherwise instructed.
The preparation of embodiment 1HMB27633 microbial bacterial agents
It carries out in accordance with the following steps：
(1) actication of culture：By be stored in -80 DEG C bacterial strain HMB27633 (Bacillus amyloliquefaciens strain HMB27633 oneself
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center's (preservation address within 29th in September in 2015：Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of city Chaoyang District North Star West Road 1), deposit number is CGMCC No.11454) in LB
(its constituent and its weight ratio are plating medium：Tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder
15g, water 1000mL) on activated (30 DEG C), picking single bacterium colony is in LB slant mediums (its constituent and its weight ratio
For：Tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL) on cultivated 12 hours at 30 DEG C,
The bacterial strain that must be activated；
(2) preparation of seed liquor：Making LB liquid medium according to a conventional method, (its constituent and its weight ratio are：Pancreas
Peptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL), it is packed into LB liquid medium in 250mL triangular flasks
100mL, high pressure moist heat sterilization access activated bacterial strain in an oese step (1) after temperature drops to room temperature, in every bottle,
Shaken cultivation is carried out under conditions of 30 DEG C, shaking speed 190rpm 12 hours, obtain seed liquor；
(3) preparation of corn flour soybean powder medium：According to weight percent 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 to get 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
It is DEG C spare；
(4) fermented and cultured：Into every bottle of corn flour soybean powder medium 200mL obtained by step (3) obtained by inoculation step (2)
Seed liquor 2mL；Fermented and cultured 36 hours is carried out under the conditions of 30 DEG C, shaking speed 180rpm, later every 30 minutes from triangle
Bottle in sampling carry out microscopy, in the visual field brood cell and total thalline number count, and calculate brood cell lead (brood cell lead (%)=at
Ripe brood cell's number/(ripe brood cell's number+thalline number) × 100)；Brood cell, which leads, stops fermented and cultured when reaching 90%；Common fermentation culture 48
Hour, obtain the liquid preparation of bacillus amyloliquefaciens HMB27633.
Antagonism tests of the 2 bacillus amyloliquefaciens HMB27633 of embodiment to tomato gray mould bacterium
(1) test period and place：In early August, 2013 is in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie plant
It is carried out in biological control of diseases laboratory.
(2) test method：
(1) for examination tomato gray mould bacterium source：Tomato gray mould bacterium BC-1 bacterial strains pick up from Hebei province Baoding Rongcheng County Dong Niudong
Village village tomato disease fruit, isolates and purifies through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, Agricultural University Of Hebei's Plant Protection
Department of plant pathology of institute is accredited as the pathogen of Botrytis cinerea (Botrytis cinerea), and Pathogenic Tests show as High pathogenicity.
(2) tablet face-off experiment：
Tomato gray mould bacterium BC-1 is activated on PDA plate first, in colony edge region after cultivating 4 days, uses card punchBacterium piece is made in punching, by the switching of tomato gray mould bacterium bacterium piece in another PDA plate center, then by 1 step of embodiment
Suddenly the bacillus amyloliquefaciens HMB27633 points activated in (1) are connected on away from 2.0 centimeters of indicator bacteria bacterium piece, if blank control is (no
Point connects the tomato gray mould bacterium growing state of HMB27633 bacterial strains).25 DEG C of constant temperature incubations wait for that blank control will cover with entire culture
When ware, control increment (colony radius) and (the inhibition life after inoculation HMB27633 of processing increment of tomato gray mould bacterium are measured
Major radius), antagonism bacteriostasis rate (calculation formula：Bacteriostasis rate (%)=(control increment-processing increment)/control life
Long amount × 100) it indicates.
As a result the bacillus amyloliquefaciens HMB27633 of the present invention that (is shown in Table 1) is to the inhibiting rate of tomato gray mould bacterium up to 89.47%；
12.0 millimeters of transparent antibacterial bandwidth；Illustrate that bacillus amyloliquefaciens HMB27633 there is apparent inhibition to make tomato gray mould bacterium
With good to the control effect of graw mold of tomato.
Antagonism test result of the 1 HMB27633 bacterial strains of table to tomato gray mould bacterium
| Strain name | Compare increment (mm) | Handle increment (mm) | Bacteriostasis rate (%) |
| HMB27633 | 38.0 | 4.0 | 89.47 |
3 bacillus amyloliquefaciens HMB27633 of embodiment is to graw mold of tomato control effect contrast test
(1) test process：
(1) microbial bacterial agent：HMB27633 liquid preparations prepared by embodiment 1；It is diluted with water 50 times.
(2) chemical agent：Fluoxastrobin suspending agent (250 grams per liter) (Syngenta Co., Ltd of Britain)；It is diluted with water 500 times.
(3) blank control：Clear water
(2) test method：This experiment in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's disease flocking biocontrol laboratory into
Row.Tomato seedling is cultivated in 50 hole seedlings nursing plates, and (tomato L-402, Liaoning gardening seedling company, Liaoning Academy of Agricultural Sciences's gardening are ground
Study carefully and produced), it is transplanted to when growing 2 compound leaves in the flowerpot of 20 centimetres of diameter, per 1 seedling of basin.Plant strain growth is to 7-8 piece compound leaves
When, select leaf age consistent, blade of the same size first uses aseptic water washing leaf table, after aseptic filter paper suck dry moisture, then is implementing
Rear (suspension mycetome 10 is diluted with water in 1 gained of example7Cfu/mL one is dipped in bacillus amyloliquefaciens HMB27633 microbial inoculums)
Under, make leaf surfaces fully medicine, be then put in the culture dish for being covered with dual-layer sterilization filter paper, while accessing tomato gray mould bacterium
BC-1 bacterium disks (diameter 6mm), moisturizing culture.The blade handled using unused HMB27633 microbial inoculums is blank control.Wait for blank control
Lesion diameter is investigated after the onset of fully, calculates control effect.
As a result the excised leaf that (is shown in Table 2) life survey the result shows that, bacillus amyloliquefaciens HMB27633 is to graw mold of tomato
Preventive effect is 85.83%, and the preventive effect of chemical agent is 71.67%.Illustrate bacillus amyloliquefaciens HMB27633 of the present invention and its micro-
Bacteria agent is significantly better than the control effect of graw mold of tomato the control effect of chemical agent.
Comparative test results of the 2 bacillus amyloliquefaciens HMB27633 of table to graw mold of tomato preventive effect
| Processing | Lesion diameter (mm) | Preventive effect (%) |
| HMB27633 liquid preparations | 3.4c | 85.83 |
| Chemical agent | 6.8b | 71.67 |
| Blank control | 24.0a | -- |
Contrast tests of the 4 bacillus amyloliquefaciens HMB27633 of embodiment to graw mold of tomato control effect
(1) test process：
(1) microbial bacterial agent：HMB27633 liquid preparations prepared by embodiment 1；It is diluted with water 50 times.
(2) chemical agent：Fluoxastrobin suspending agent (250 grams per liter) (Syngenta Co., Ltd of Britain)；It is diluted with water 500 times.
(3) blank control：Clear water
(2) test method：In December, 2013 in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's artificial climate room into
Row.Tomato seedling (tomato L-402, Liaoning gardening seedling company, Gardening Inst, Liaoning Agricultural Academy are cultivated in heliogreenhouse
Production), it waits for that tomato growth goes out 8 compound leaves, moves to temperature and humidity control culturing room, choose tomato seedling of the same size, embodiment 1 is made
Standby 50 times of water diluents of HMB27633 liquid preparations are uniformly sprayed on tomato seedling, 25 DEG C culture 24 hours after in every leaflet
Upper access tomato gray mould bacterium bacterium disk (cultivating 3-5d, diameter 6mm at 25 DEG C on PDA plate), moisturizing culture.If clear water and chemical drugs
Agent compares, and investigates morbidity after 3d as a result, measuring each lesion diameter (A, B) using cross mensuration, calculates lesion area and prevents
Control effect.
Lesion area (mm2)=π × A × B/4
Control effect (%)=(control lesion area-processing lesion area)/control lesion area × 100
As a result the live body that (is shown in Table 3) life survey the result shows that, bacillus amyloliquefaciens HMB27633 also significantly inhibits tomato gray mould
Extension of the bacterium on tomato leaf reaches 82.20% to the control effect of graw mold of tomato, and the preventive effect of chemical agent is
73.02%.Illustrate that bacillus amyloliquefaciens HMB27633 and its microbial bacterial agent are significantly better than graw mold of tomato control effect
The control effect of chemical agent.
Comparative test results of the 3 bacillus amyloliquefaciens HMB27633 of table to graw mold of tomato preventive effect
| Processing | Lesion area (mm2) | Preventive effect (%) |
| HMB27633 liquid preparations | 20.07c | 82.20 |
| Chemical agent | 30.42b | 73.02 |
| Blank control | 112.76a | -- |
Contrast tests of the 5 bacillus amyloliquefaciens HMB27633 of embodiment to graw mold of tomato control effect
(1) test process：
(1) microbial bacterial agent：HMB27633 liquid preparations prepared by embodiment 1, are diluted with water 50 times.
(2) chemical agent：Fluoxastrobin suspending agent (250 grams per liter) (Syngenta Co., Ltd of Britain)；It is diluted with water 500 times.
(3) blank control：Clear water
(2) test method：2~June in 2014 Hebei province Mancheng County of Baoding contest village Sun Mao dragon tomato greenhouses into
Row.Start tomato leaf portion gray mold moderate generation in entire canopy when experiment, sick leaf is extractd before dispenser.Per three row of cell, four weights
It is multiple, random district's groups arrangement.Using the bacillus amyloliquefaciens HMB27633 prepared by knapsack electric sprayer inoculation embodiment 1
Microbial inoculum (is diluted, mycetome 10 with water7cfu/mL)；Separately set comparison medicament and blank control.Dispenser for the first time sprays again after 7 days
1 time, the single plant disease number of sheets of each processing cell of investigation in the 7th day after secondary dispenser, and calculate preventive effect.
As a result the single plant disease of graw mold of tomato after the microbial inoculum processing for the bacillus amyloliquefaciens HMB27633 of the present invention that (is shown in Table 4)
The number of sheets is substantially less than blank control and medicament control, and preventive effect reaches 92.93%, illustrates Bacillus amyloliquefaciens strain bacterial strain
HMB27633 is demonstrated by preferable control effect to graw mold of tomato.
Comparative test results of the 4 bacillus amyloliquefaciens HMB27633 of table to graw mold of tomato preventive effect
| Processing | The single plant disease number of sheets (piece) | Preventive effect (%) |
| HMB27633 liquid preparations | 0.33c | 92.93 |
| Chemical agent | 1.33b | 71.52 |
| Blank control | 4.67a | --- |
Claims (5)
1. a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain HMB27633, oneself was in 2015 9
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC on the 29th
No.11454。
2. the microbial bacterial agent produced using bacillus amyloliquefaciens described in claim 1, it is characterised in that its active constituent
For HMB27633 thalline.
3. microbial bacterial agent according to claim 2, it is characterised in that the microbial bacterial agent is liquid preparation.
4. applications of the bacillus amyloliquefaciens HMB27633 described in claim 1 on prevention graw mold of tomato.
5. application of the microbial bacterial agent on prevention graw mold of tomato described in claim 2.
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| CN106337032A (en) * | 2016-10-08 | 2017-01-18 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens for preventing botrytis cinerea and application thereof |
| CN106434454A (en) * | 2016-10-08 | 2017-02-22 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens and inoculant for preventing and treating tomato botrytis cinerea |
| CN106635921B (en) * | 2017-01-17 | 2019-09-17 | 陈刘军 | Bacillus amyloliquefaciens Y15 and its application |
| CN106676047B (en) * | 2017-02-23 | 2019-07-30 | 邢台学院 | The preparation method and application of bacillus amyloliquefaciens HM-6 and its microbial inoculum |
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| CN104498386A (en) * | 2014-11-25 | 2015-04-08 | 山西农业大学 | Preparation method and applications of wild jujube endophytic bacillus amyloliquefaciens new strain SZ23 and fermentation broth |
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