CN106434454A - Bacillus amyloliquefaciens and inoculant for preventing and treating tomato botrytis cinerea - Google Patents
Bacillus amyloliquefaciens and inoculant for preventing and treating tomato botrytis cinerea Download PDFInfo
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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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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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 invention discloses a bacillus amyloliquefaciens strain HMB27636 for preventing and treating tomato botrytis cinerea. The strain is preserved in General Microbiological Culture Collection Center of China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No.13038. The invention further discloses a microbial inoculant produced by the strain, and a preparation method thereof. The bacillus amyloliquefaciens strain HMB27636 has good prevention and treatment effect on the tomato botrytis cinerea, and the average prevention effect is more than 80.0%; secondarily, HMB27636 has strong tomato botrytis cinerea specialization, and cannot generate drug resistance easily; in addition, the microbial inoculant is safe to human and livestocks, and free from environmental pollution; and the preparation method of the microbial inoculant is simple and low in cost.
Description
Technical field
The invention belongs to field of biological control, be specifically related to a kind of solution starch gemma bar for preventing and treating graw mold of tomato
Bacterium；Further relate to the microbial bacterial agent containing this bacillus amyloliquefaciens, and their purposes on preventing and treating graw mold of tomato.
Background technology
Graw mold of tomato is that the one being caused by the pathogen of Botrytis cinerea (Botrytis cinerea) of Deuteromycotina is important
Disease.This disease main harm fruit, causes fruit rot, it is possible to harm leaf, stem and flower, the production loss causing is up to 20%
～50%；Serious threat tomato in greenhouse produces.
The method of preventing and treating graw mold of tomato mainly has：(1) disease-resistant variety is cultivated：This is the most cost-effective method, but
It is owing to anti-source material is limited, and due to biological common evolutionary, the biological strain of new pathogen constantly occurs, therefore trains
Educate disease-resistant variety approach effect limited；(2) chemical prevention：This is the Main Means of preventing and treating graw mold of tomato, but uses chemistry
There is the resistance to the action of a drug and lose the problem with environmental pollution, the health of serious harm people and animals in chemical control；(3) biological control：Due to tool
Have that specialization is strong, be not likely to produce the advantage such as the resistance to the action of a drug, lasting medicine, be increasingly subject to the favor of people.
Bacillus (Bacillius sp) has distribution cultivation wide, easily separated, can produce the stronger brood cell of resistance, storage
Tibetan phase length and the advantage such as easy to use, become a kind of preferable Biocontrol microorganism.It is currently used for preventing and treating the gemma of graw mold of tomato
Bacillus mainly has：Bacillus licheniformis (CN104232499A), bacillus pumilus (CN101928681A), solution starch gemma bar
Bacterium (CN103173397A, CN104531559A, CN105176894A, CN104762223A, CN102604864A,
CN105199996A, CN104498386A), Bacillus circulans (CN103952352A), bacillus subtilis
(CN1934241A, CN103074271A, CN101993836A, CN101148650A), Brevibacillus laterosporus
And deep brown bacillus (CN104946567A) etc. (CN104480046A).From the above, it can be seen that pathogen is had by screening
The bacillus of inhibitory action is one of the most effectual way prevented and treated about plant disease.
Content of the invention
Present invention aim at providing a kind of Bacillus amyloliquefaciens strain for preventing and treating graw mold of tomato, this bacterial strain has
Have the advantages such as efficient, fungicidal spectrum is wide.
Another object of the present invention is to provide a kind of microbial bacterial agent.
The present invention the 3rd purpose is to provide the preparation method of mentioned microorganism microbial inoculum.
The present invention the 4th purpose is to provide purposes on preventing and treating graw mold of tomato for the above-mentioned bacillus amyloliquefaciens.
The present invention the 5th purpose is to provide purposes on preventing and treating graw mold of tomato for the mentioned microorganism microbial inoculum.
The present invention is achieved through the following technical solutions：
The invention provides a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain
HMB27636, oneself was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (protects on September 26th, 2016
Hiding address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica), deposit number is CGMCC
No.13038.
Utilizing the microbial bacterial agent that above-mentioned bacillus amyloliquefaciens HMB27636 produces, its active component is for solving starch gemma
Bacillus HMB27636 thalline.
Mentioned microorganism microbial inoculum can be liquid preparation.
The preparation method of mentioned microorganism microbial inoculum, comprises the steps：
(1) actication of culture：Activating the HMB27636 bacterial strain of Cord blood on LB plating medium, picking list bacterium colony exists
On LB slant medium, cultivate 10～16 hours at 25～35 DEG C, obtain the bacterial strain of activation；
(2) prepared by seed liquor：The bacterial strain that activates by aseptic oese scraping one oese step (1) is simultaneously inoculated into
In 100mL LB fluid nutrient medium, 25～35 DEG C, shaking speed be 150～220rpm under the conditions of cultivate 10～16 hours,
Seed liquor；
(3) fermented and cultured：According to the ratio that volume ratio is 1～3%, the seed liquor of step (2) is inoculated into corn flour soya bean
In powder culture medium (pH value is 7.2), 25～35 DEG C, shaking speed be 150～220rpm under conditions of fermented and cultured 40～
50h, obtains zymotic fluid；
(4) detect thalline and brood cell's quantity in zymotic fluid, treat that ripe brood cell in zymotic fluid accounts for brood cell and thalline sum
Stopping fermented and cultured when 90%, gained is the liquid preparation of HMB27636 bacterial strain.
LB plating medium described in above-mentioned preparation method, LB slant medium or LB fluid nutrient medium are all according to routine
Prepared by method.
The constituent of LB plating medium or LB slant medium described in above-mentioned preparation method step (1) and weight thereof
Ratio is：Tryptone 8～12g, yeast extract 4～6g, sodium chloride 4～6g, agar powder 12～18g, water 1000mL.
Constituent and the weight ratio thereof of LB fluid nutrient medium described in above-mentioned preparation method step (2) are：Tryptone 8
～12g, yeast extract 4～6g, sodium chloride 4～6g, water 1000mL.
Corn flour soybean powder medium described in above-mentioned preparation method step (3), its constituent and weight percent thereof
Ratio is：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.
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 mixes, and adds water, and regulation pH stirs.
Mentioned microorganism microbial inoculum, the viable count of its bacillus amyloliquefaciens HMB27636 is more than 21.0 × 108cfu/mL.
Application on preventing and treating graw mold of tomato for the above-mentioned bacillus amyloliquefaciens HMB27636.
Application on preventing and treating graw mold of tomato for the mentioned microorganism microbial inoculum.
The using method of mentioned microorganism microbial inoculum：With water, above-mentioned gained microbial bacterial agent being diluted to viable bacteria body number is
107Cfu/mL, carries out foliar spray in graw mold of tomato premorbid.
The screening separation process of HMB27636 bacterial strain of the present invention：
In April, 2013,5 collections from cotton field, Jingzhou City of Hubei Province of Hebei Prov. Academy of Agricultural ＆. Forest Sciences's Plant Protection Institute were native
Sample, weighs 1g and is put in the sterilizing triangular flask of 250mL after mixing, add 100mL sterilized water, be put on shaking table, and 170r/min shakes
Swing 30min, stand 2h, take supernatant 10mL and add in 50mL sterile centrifugation tube, 80 DEG C of waters bath with thermostatic control 30 minutes, then take
1mL adds sterilized water 9mL, 10mL10-3Soil supension is diluted to 10 by times edaphon suspension then-4、10-5、10-6Times
Dilution；Taking each concentration microorganism suspension 200 μ L to be applied on LB culture medium flat plate, each concentration is repeated 3 times, and trains at 30 DEG C of constant temperature
Support 1d～3d, carry out separation and the purifying of bacterium.And with graw mold of tomato as target, by flat board face-off method, Leaf method,
Pot experiment method carries out the screening of biocontrol microorganisms.Result therefrom filters out a bacterium to graw mold of tomato with obvious prevention effect
Strain, names as HMB27636.
The taxonomic identification of HMB27636 bacterial strain：
(1) identification by morphological characters
It is shaft-like for cultivating thalline on LB culture medium, produces brood cell after cultivating 10h, and raw in brood cell, oval, cyst is not swollen
Greatly, acid-fast stain is negative, without parasporal crystal, can move, flagellum Zhousheng.On nutrient agar panel, the light breast of Initial stage of culture bacterium colony
White, purulence shape, circular, neat in edge, bacterium colony swells 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；Line on nutrient agar slopes is cultivated, linear；Static gas wave refrigerator in liquid medium within, table
Face forms white mycoderm.These morphological features with《Common bacteria system identification handbook》(the elegant pearls in east etc. are write. Science Press
.2001 year) described in bacillus morphological feature basically identical, tentatively judge that bacterial strain HMB27636 belongs to bacillus.
(2) 16S rDNA Sequence Identification is utilized to classify
With the genomic DNA of HMB27636 as template, enter performing PCR amplification with F27 and R1492 to 16S rDNA for primer,
Described primer sequence is：
F27：5’-AGAGTTTGATCATGGCTCAG-3’(SEQ ID No:1)；
R1492：5’-GGCTACCTTGTTACGACTT-3’(SEQ ID No:2).
The reaction system of PCR 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 genomic DNA 50ng of HMB27636；ddH2O
Complement 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 circulations；72℃
10min.Gained pcr amplification product is carried out gel electrophoresis, delivers the order-checking of Shanghai Sheng Gong bioengineering Co., Ltd, obtain
The 16S rDNA sequence of HMB27636 is (see SEQ ID No:3).By the 16S rDNA sequence of gained HMB27636 in Genbank
Carrying out tetraploid rice, results strain HMB27636 reaches 99% with the 16S rDNA homology of bacillus；Constructing system
Grow tree (see Fig. 1), together with HMB27636 is aggregated to bacillus, illustrate that HMB27636 belongs to bacillus
(Bacillus).
(3) gyrB gene order is utilized to identify classification
It with HMB27636 genomic DNA as template, with bacillus gyrB gene degenerate primer gyrB-F and gyrB-R is
Primer enters performing PCR amplification, obtains pcr amplification product；The sequence of wherein said gyrB-F and gyrB-R primer is：
gyrB-F：5’-TTGRCGGHRGYGGHTATAAAGT-3’(SEQ ID No:4)；
gyrB-R：5’-TCCDCCSTCAGARTCWCCCTC-3’(SEQ ID No:5).
The reaction system of the PCR of gyrB is 50 μ L:10×PCR Buffer(Mg2+)5μL；dNTP Mixture(2.5mM)5
μL；Taq(5U/μL)1μL；GyrB-F (10 μm of ol/L) 1 μ L, gyrB-R (10 μm of ol/L) 1 μ L；HMB27636 genomic 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 are followed
Ring；72℃10min.Amplified production is delivered the order-checking of Shanghai Sheng Gong bioengineering Co., Ltd, obtains the gyrB of HMB27636 bacterial strain
Gene order is (see SEQ ID No:6).The gyrB gene order of HMB27636 bacterial strain is carried out homology ratio in Genbank
Relatively, it is found that HMB27636 is the highest with the gyrB gene homology of bacillus amyloliquefaciens, 98.25% is reached；Simultaneously
Utilize MEGA software building phylogenetic tree (see Fig. 2), together with HMB27636 bacterial strain is aggregated to bacillus amyloliquefaciens, explanation
HMB27636 is bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and is a new strains.
The result of comprehensive above morphological feature, 16S rDNA and gyrB gene homology comparative analysis, it is known that
HMB27636 belongs to bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and and existing bacillus bacterium
Strain is different, is a new Bacillus amyloliquefaciens strain.
The present invention has the advantage that and beneficial effect：(1) present invention provides one efficiently for preventing and treating graw mold of tomato
Microorganism, opens an effective controlling way；(2) graw mold of tomato is prevented by bacillus amyloliquefaciens HMB27636 of the present invention
Controlling effect good, average preventive effect is more than 80.0%；(3) microbial bacterial agent of the present invention is to people, animal safety, does not has environmental pollution；(4)
Utilize bacillus amyloliquefaciens HMB27636 of the present invention preventing and treating graw mold of tomato specialization strong, be not likely to produce the resistance to the action of a drug；(5) this
Bright microbial bacterial agent preparation method is simple, low cost, use are simple.
Brief description
Fig. 1. according to 16S rDNA sequence obtain HMB27636 bacterial strain systematic growth tree graph.
Fig. 2. according to gyrB gene order obtain HMB27636 bacterial strain systematic growth tree graph.
Detailed description of the invention
Clearly to explain further the present invention with specific embodiment below, but constitute never in any form to the present invention
Restriction.Experimental technique in following embodiment, if no special instructions, is conventional method；Percentage in following embodiment contains
Amount, if no special instructions, is weight percentage.
The preparation of embodiment 1HMB27636 microbial bacterial agent
Carry out in accordance with the following steps：
(1) actication of culture：By the Bacillus amyloliquefaciens strain HMB27636 that is stored in-80 DEG C, (oneself is September 26 in 2016
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.13038)
At LB plating medium, ((its constituent and weight ratio thereof are：Tryptone 10g, yeast extract 5g, sodium chloride 5g, agar
Powder 15g, water 1000mL)) on carry out activating (30 DEG C), picking list bacterium colony is in LB slant medium ((its constituent and weight thereof
Ratio is：Tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL)) on cultivate at 30 DEG C 12 little
When, obtain the bacterial strain of activation；
(2) preparation of seed liquor：LB fluid nutrient medium ((its constituent and weight ratio thereof is loaded in 250mL triangular flask
For：Tryptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL)) 100mL, high pressure moist heat sterilization, treat that temperature drops to
After room temperature, every bottle is accessed the bacterial strain having activated in an oese step (1), 30 DEG C, shaking speed 190rpm under conditions of
Carry out shaken cultivation 12 hours, obtain 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 obtains corn flour soybean powder medium；It is sub-packed in
In 500mL triangular flask, every bottle of 200mL；Carry out sterilizing 30 minutes to corn flour soybean powder medium at 121 DEG C, then cool to 30
DEG C standby；
(4) fermented and cultured：Inoculation step (2) gained in step (3) gained every bottle corn flour soybean powder medium 200mL
Seed liquor 2mL；30 DEG C, carry out fermented and cultured 36 hours under the conditions of shaking speed 180rpm, later every 30 minutes from three
In the bottle of angle sampling carry out microscopy, the brood cell in the visual field and total thalline number are counted, and calculate brood cell lead (brood cell lead (%)=
Ripe brood cell's number/(ripe brood cell's number+thalline number) × 100)；Brood cell leads stopping fermented and cultured when reaching 90%；Fermented and cultured altogether
48 hours, obtain the liquid preparation of bacillus amyloliquefaciens HMB27636.
The antagonism test to tomato gray mould bacterium for the embodiment 2 bacillus amyloliquefaciens HMB27636
(1) for examination botrytis cinerea source：Botrytis cinerea BC-1 bacterial strain picks up from east, Rongcheng County, Baoding, Hebei province
The sick fruit of Niu Dongzhuan village tomato, isolated and purified through Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, Agricultural University Of Hebei is accredited as
The pathogen of Botrytis cinerea (Botrytis cinerea), Pathogenic Tests shows as High pathogenicity.
(2) test method：
This is tested in early April, 2014 at Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's biocontrol of plant disease
Carry out in laboratory.First by botrytis cinerea BC-1 on PDA plate activation culture 4 days, then use card punchMake bacterium piece in the punching of colony edge region, then transfer botrytis cinerea BC-1 bacterium piece at another
PDA plate central authorities, then be connected on the bacillus amyloliquefaciens HMB27636 point after embodiment 1 step (1) activation away from indicator bacteria bacterium
Piece 2.0 centimeters, if blank (do not put and connect HMB27636 bacterial strain).Incubated at 25 DEG C, treat that blank will cover with
During whole culture dish, the comparison increment (colony radius) of measurement tomato gray mould bacterium and process increment are (after inoculation HMB27636
Developing restraint radius), antagonism bacteriostasis rate represents.The computing formula of bacteriostasis rate is：
Bacteriostasis rate (%)=(comparison increment-process increment)/comparison increment × 100).
The inhibiting rate to tomato gray mould bacterium for result (being shown in Table 1) the bacillus amyloliquefaciens HMB27636 of the present invention reaches 68.2%；
Transparent antibacterial bandwidth 2.0 millimeters, bacterial growth reaches 11 millimeters, and competition for space and nutrient competition effect are very strong；Illustrate to solve starch
Bacillus HMB27636 has obvious inhibitory action to tomato gray mould bacterium, has the Biocontrol Potential of preventing and treating graw mold of tomato.
The antagonism test result to botrytis cinerea for the table 1HMB27636 bacterial strain
Embodiment 3 bacillus amyloliquefaciens HMB27636 is to graw mold of tomato preventive effect contrast test
(1) test process：
(1) microbial bacterial agent：The HMB27636 liquid preparation of embodiment 1 preparation, dilute with water 50 times.
(2) chemical agent：Fluoxastrobin suspending agent (250 grams per liter) (Syngenta Co., Ltd of Britain), dilute with water 500 times.
(3) blank：Clear water
(2) test method：This test is entered in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's disease flocking biocontrol laboratory
OK.Cultivate the tomato seedling of Ao Saite (968) (being purchased from Beijing Shuo Yuan seeds company) in 50 hole seedlings nursing plates, when growing 2 compound leaves
It is transplanted in the flowerpot of diameter 20 centimetres, 1 seedling of every basin.When plant strain growth is to 7-8 piece compound leaf, select leaf age consistent, size one
The blade causing, first uses aseptic water washing leaf table, then uses aseptic filter paper suck dry moisture, then the solution starch bud in embodiment 1 preparation
Spore bacillus HMB27636 microbial inoculum water diluent (mycetome 107Cfu/mL) dip in, the medicine that makes leaf surfaces fully, then put
In the culture dish being covered with dual-layer sterilization filter paper, being concurrently accessed botrytis cinerea BC-1 bacterium dish (diameter 6mm), moisturizing is cultivated.
With unused HMB27636 microbial inoculum process blade as blank.Treat that blank is fully fallen ill " Invest, Then Investigate " lesion diameter, calculate
Prevention effect.
Result (being shown in Table 2) is in the raw test of excised leaf is tested, and graw mold of tomato is had by bacillus amyloliquefaciens HMB27636
Preventive effect reach 72.77%, not notable with the preventive effect of chemical reference medicament (80.38%) difference.Bacillus amyloliquefaciens is described
HMB27636 and microbial bacterial agent thereof have good prevention effect to graw mold of tomato.
Table 2 bacillus amyloliquefaciens HMB27636 is to graw mold of tomato preventive effect comparative test result
| Process | Lesion diameter (mm) | Preventive effect (%) |
| HMB27636 liquid preparation | 7.2b | 72.77 |
| Chemical agent | 5.2b | 80.38 |
| Blank | 26.5a | -- |
Embodiment 4 bacillus amyloliquefaciens HMB27636 is to graw mold of tomato preventive effect contrast test
(1) test process：
(1) microbial bacterial agent：The HMB27636 liquid preparation of embodiment 1 preparation；Dilute with water 50 times.
(2) chemical agent：Fluoxastrobin suspending agent (250 grams per liter) (Syngenta Co., Ltd of Britain)；Dilute with water 500 times.
(3) blank：Clear water
(2) test method：
In December, 2013 is carried out in Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie's artificial climate indoor.At heliogreenhouse
Cultivate the tomato seedling of Ao Saite (968) (being purchased from Beijing Shuo Yuan seeds company), treat that tomato growth goes out 8 compound leaves, move to temperature control control
Wet culturing room, chooses tomato seedling of the same size, and the 50 times of water diluents of HMB27636 liquid preparation preparing embodiment 1 are uniform
It is sprayed on tomato seedling, cultivate 24 hours at 25 DEG C, on every leaflet, then access botrytis cinerea BC-1 bacterium dish (PDA
3-5d, diameter 6mm is cultivated at 25 DEG C on flat board), 3d is cultivated in moisturizing.If clear water blank and Chemical treatment.Investigation is sent out
Sick result, uses cross mensuration to measure each lesion diameter (A, B), calculates lesion area and prevention effect.
Lesion area (mm2)=π × A × B/4；
Prevention effect (%)=(comparison lesion area-process lesion area)/comparison lesion area × 100.
The comparative test result to graw mold of tomato preventive effect for the table 3 bacillus amyloliquefaciens HMB27636
| Process | Lesion area (mm2) | Preventive effect (%) |
| HMB27636 liquid preparation | 18.96c | 83.19 |
| Chemical agent | 30.42b | 73.02 |
| Blank | 112.76a | -- |
Result (being shown in Table 3) in the raw test of live body is tested, the scab face that bacillus amyloliquefaciens HMB27636 of the present invention is processed
The long-pending lesion area significantly less than Chemical treatment and blank, to pressing down of extension on tomato leaf for the tomato gray mould bacterium
Make of significantly；The preventive effect to graw mold of tomato for the bacillus amyloliquefaciens HMB27636 microbial inoculum of the present invention is 83.19%, also substantially
Preventive effect higher than chemical agent 73.02%.Illustrate bacillus amyloliquefaciens HMB27636 and microbial bacterial agent thereof to tomato gray mould
Sick prevention effect is good.
The field comparison test to graw mold of tomato preventive effect for the embodiment 5 bacillus amyloliquefaciens HMB27636
(1) test process：
(1) microbial bacterial agent：The HMB27636 liquid preparation of embodiment 1 preparation, dilute with water 50 times.
(2) chemical agent：Fluoxastrobin suspending agent (250 grams per liter) (Syngenta Co., Ltd of Britain)；Dilute with water 500 times.
(3) blank：Clear water
(2) test method：In February, 2014 to June enters at contest village, Mancheng County of Baoding, Hebei province Sun Mao dragon tomato booth
OK.Start gray mold moderate generation in tomato leaf portion in whole canopy during test, before dispenser, extract sick leaf.Every cell 3 row, repeats for 4 times,
The group arrangement of random district.Use the bacillus amyloliquefaciens HMB27636 liquid of knapsack electric sprayer inoculation embodiment 1 preparation
50 times of water diluent (mycetomes 10 of preparation7cfu/mL)；Separately set Chemical treatment and blank.After dispenser first 7 days
Spraying 1 time, the sick number of sheets of each individual plant processing cell is investigated in the 7th day after dispenser for the second time, and calculates preventive effect again.
After the microbial inoculum process of result (being shown in Table 4) bacillus amyloliquefaciens HMB27636 of the present invention, the individual plant of graw mold of tomato is sick
The number of sheets is substantially less than blank and Chemical treatment, and its preventive effect reaches 78.72%, higher than the preventive effect of chemical agent, explanation
Bacillus amyloliquefaciens strain HMB27636 of the present invention and microbial inoculum thereof are good to the prevention effect of graw mold of tomato.
Table 4 bacillus amyloliquefaciens HMB27636 is to graw mold of tomato preventive effect comparative test result
| Process | The sick number of sheets (piece) of individual plant | Preventive effect (%) |
| HMB27636 liquid preparation | 1.0c | 78.72 |
| Chemical agent | 1.33b | 71.52 |
| Blank | 4.67a | -- |
Claims (9)
1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain HMB27636, oneself was in 2016 9
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 26th, and deposit number is CGMCC
No.13038.
2. utilize the microbial bacterial agent that the bacillus amyloliquefaciens HMB27636 described in claim 1 produces, it is characterised in that its
Active component is bacillus amyloliquefaciens HMB27636 thalline.
3. microbial bacterial agent according to claim 2, it is characterised in that be liquid preparation.
4. the preparation method of the microbial bacterial agent described in claim 3, comprises the steps：
(1) activating the HMB27636 bacterial strain of Cord blood on LB plating medium, picking list bacterium colony is at LB slant medium
On, cultivate 10～16 hours at 25～35 DEG C, obtain the bacterial strain of activation；
(2) bacterial strain that activates by aseptic oese scraping one oese step (1) is simultaneously inoculated into 100mLLB fluid nutrient medium
In, 25～35 DEG C, shaking speed be 150～220rpm under the conditions of cultivate 10～16 hours, obtain seed liquor；
(3) according to the ratio that volume ratio is 1～3%, the seed liquor of step (2) is inoculated into corn flour soybean powder medium (pH value
Be 7.2) in, 25～35 DEG C, shaking speed be 150～220rpm under conditions of fermented and cultured 40～50h, obtain zymotic fluid；
(4) detect thalline and brood cell's quantity in zymotic fluid, treat that in zymotic fluid, ripe brood cell accounts for the 90% of brood cell and thalline sum
When stop fermented and cultured, gained is the liquid preparation of HMB27636 bacterial strain.
5. preparation method according to claim 4, it is characterised in that described in its step (1), LB plating medium or LB are oblique
The constituent of face culture medium and weight ratio thereof are：Tryptone 8～12g, yeast extract 4～6g, sodium chloride 4～6g, fine jade
Cosmetics 12～18g, water 1000mL.
6. preparation method according to claim 4, it is characterised in that the composition of LB fluid nutrient medium described in its step (2)
Composition and weight ratio thereof are：Tryptone 8～12g, yeast extract 4～6g, sodium chloride 4～6g, water 1000mL.
7. preparation method according to claim 4, it is characterised in that the corn flour analysis for soybean powder described in its step (3) is cultivated
The constituent of base and percentage by weight thereof are：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.
8. application on preventing and treating graw mold of tomato for the bacillus amyloliquefaciens HMB27636 described in claim 1.
9. application on preventing and treating graw mold of tomato for the microbial bacterial agent described in claim 2.
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| CN108077411A (en) * | 2017-12-20 | 2018-05-29 | 山西省农业科学院农产品贮藏保鲜研究所 | The preparation method and its application method of bacillus amyloliquefaciens Y-3 bio-preservatives |
| CN108531429A (en) * | 2018-04-27 | 2018-09-14 | 山东农业大学 | It is a kind of to produce protease, the Xiamen bacillus of anti-botrytis cinerea and its application |
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| CN105176894A (en) * | 2015-11-03 | 2015-12-23 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens for controlling gray mold of tomato and microbial inoculant thereof |
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| CN105176894A (en) * | 2015-11-03 | 2015-12-23 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens for controlling gray mold of tomato and microbial inoculant thereof |
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| CN108531429A (en) * | 2018-04-27 | 2018-09-14 | 山东农业大学 | It is a kind of to produce protease, the Xiamen bacillus of anti-botrytis cinerea and its application |
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