CN106479929B - Bacillus amyloliquefaciens and its application in the prevention and treatment of numb Chinese yam root rot - Google Patents
Bacillus amyloliquefaciens and its application in the prevention and treatment of numb Chinese yam root rot Download PDFInfo
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Abstract
The invention belongs to the production of microbial bacterial agent, particularly relate to bacillus amyloliquefaciens and its application in the prevention and treatment of numb Chinese yam root rot.Be bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12821 is inoculated in containing carbon source, nitrogen source and necessary nutrient without bacteria fermentation culture medium, carry out aerobic fementation and the liquid microbial microbial inoculum containing above-mentioned viable bacteria be made.The present invention has filled up there is no bacillus amyloliquefaciens for technological gap that numb Chinese yam root rot prevents and treats at present, the advantages such as all technical with prepared bacillus amyloliquefaciens microbial inoculum meets the requirement of GB20287-2006, and good to numb Chinese yam root rot control efficiency and effect of increasing production and numb Chinese yam stripe shape is straight, coarse, white color, abnormal rate are low.
Description
Technical field
The invention belongs to the production of microbial bacterial agent, particularly relates to bacillus amyloliquefaciens and its prevent in numb Chinese yam root rot
Application in controlling.
Background technique
Numb Chinese yam (Dicscorea opposite Thunb) belongs to Dioscoreaceae herbaceous perennial vine plant, and rhizome nutrition is rich
Richness has food, medicine function concurrently, has invigorating the spleen, tonifying lung, strengthening the essence and other effects.Modern research shows that the polysaccharide in numb Chinese yam, which has, to be reduced
Blood glucose, raising and the function that is immunized, prevents hyperlipidemia, adjust stomach and intestine, preventing aging is adjusted, the allantois contained in numb Chinese yam is known as
Anti-irritant object, anesthesia is calm, promotes epithelial growth, the effects of anti-inflammatory is antibacterial.Therefore numb Chinese yam is increasingly subject to domestic and international consumer
Welcome.The situation of China fiber crops Chinese yam foreign exchange earning in recent years is fine, and the price of domestic market is also promoted steadily, cultivates Chinese yam
Economic benefit is to plant wheat, 8-l0 times of corn, and cultivated area expands year by year, and only there are nearly ten thousand public affairs in Hebei province along the two sides pool Long He
Cultivated area just, has gone on industrialized development road, has become the main economic revenue source of local farmers.But with growing surface
Long-pending expansion and uneven fertilising phenomenon are got worse, and being excessively used for chemical fertilizer continues to decline soil regime, edaphon
Environment is destroyed, and occurs a large amount of problems in planting process, and pest and disease damage happens occasionally, and continuous cropping phenomenon is serious, often will cause
The underproduction or total crop failure.Root rot is one kind of numb Chinese yam continuous cropping disease, is to endanger one of numb Chinese yam production most serious disease.It is raw at present
The generation of the method reduction root rot of crop rotation and chemical bactericide disinfection potato seed is generallyd use in production, but due to soil-borne disease cause of disease
Host range is wide for bacterium, and crop rotation is hard to work sometimes.And Pesticide use can not only cause the direct damage to human health, also make
The pollution of pairs of environment, pathogen is easy to produce drug resistance after long-time service, increases difficulty of prevention and cure, forms vicious circle.With
The development of biotechnology, people start to recognize that application microbial bacterial agent is to improve soil environment, fundamentally solve crop weight
The effective measures of stubble problem.
Bacillus amyloliquefaciens are gram-positive bacterias, very high with bacillus subtilis affinity, in the life of its own
It can produce a series of metabolite in growth process.These metabolites make bacillus amyloliquefaciens that can widely inhibit true
The activity of bacterium and bacterium.The general speed of growth is fast, is plant rhizosphere soil and the intracorporal important microbe of plant surface, antimicrobial spectrum
Extensively, there is preferable bacteriostasis for many phytopathogens.
The background document that applicant retrieves includes:
1,1,000,000,000 gemma/g bacillus subtilis wettable powder " northern gardening " 2011 (01): is disclosed in 159-160 "
Effective dose to numb Chinese yam root rot is 15kg/hm2, be averaged control efficiency 70.34%, is significantly higher than comparison medicament fenaminosulf
Preventive effect (20.26%), illustrate that it has significant stimulating growth effect to numb Chinese yam absorption root, field relative control effect reaches as high as
88.65%.
2, prevention plastic tent cucumber continuous cropping disease probiotics microbial inoculum is disclosed in the patent document of Publication No. CN102382810A
Preparation method, major technique solution is that the production strain of probiotics is inoculated in containing carbon source, nitrogen source, inorganic salts
It ferments in the aseptic culture medium of water, fermentation process is carried out under conditions of temperature is 30-37 DEG C, pH value 7.2-7.6
Aerobic fermentation, production strain selects bacillus subtilis, bacillus licheniformis, shellfish thunder trichosporon cutaneum, Paecilomyces lilacinus, thin yellow
Streptomycete, bacillus polymyxa and Metarhizium anisopliae etc. mass mixings object, fermentation end products are adsorbed and fixed in turf
On probiotics microbial inoculum is made.Cucumber mycosis, root rot and verticillium wilt can effectively be prevented.
3, bacillus amyloliquefaciens cd5bc is disclosed in the patent document of Publication No. CN105907683A to capsicum sclerotium
Disease, bitter gourd wilt, cucumber fusarium axysporum, yellow scab, rice blast, rice sheath blight disease, semen viciae fabae red spot, semen viciae fabae brown spot
Pathogen bacteriostasis rate be up to 70% or more, the bacteriostasis rate of semen viciae fabae red spot, semen viciae fabae Pathogenic Bacteria Causing Brown Blotch Disease is up to
100%.
It is applicant's understanding that Different Crop is easily caught an illness, bacterium is different, and different strains are to the control efficiency of same germ
There are larger differences.Bacillus amyloliquefaciens are used for numb Chinese yam root rot prevention and treatment at present and have no that pertinent literature is reported.
Summary of the invention
The purpose of the present invention is to provide bacillus amyloliquefaciens and its applications in the prevention and treatment of numb Chinese yam root rot.
Overall technology design of the invention is:
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12821.
Strain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC in the present invention
No.12821 system applicant separates acquirement from Hebei province, Dingxing County, Baoding fiber crops Chinese yam rhizosphere soil, and applicant is in 2016
Submit within August 8 days the China Committee for Culture Collection of Microorganisms for being located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 common
The preservation of microorganism center, depositary institution abbreviation CGMCC.
The morphological feature and physiological and biochemical property of the strain are as follows:
Strain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC in the present invention
No.12821 is Gram-positive, round smooth colony, is moistened, neat in edge, raw in gemma in yellowish, is not expanded, ellipse
Circle produces acid on dextrose culture-medium, produces gas V.P reacting positive, can hydrolyze starch, not grow under anaerobic condition with 3 DEG C,
It is grown in 7% sodium chloride, G+C content is 43.5-44.9%.Thallus size is 0.7-0.8 × 2-3 μm.
Above-mentioned strain has efficient phosphorus decomposing, ability of dissolving potassium, with Sodium Tetraphenylborate Method and molybdenum antimony resistance colorimetric method measurement potassium decomposing, solution
Phosphorus ability, be as a result respectively ability of dissolving potassium be 6.13mg/L, the organic phosphorus ability of solution is 7.95mg/L, solution Phos ability is
82.79mg/L；Numb Chinese yam root is inoculated with after 30 days, after measured, bacillus amyloliquefaciens quantity has 10-100 times of increase, explanation
The bacterial strain has good colonization ability.
Application of the bacillus amyloliquefaciens in the prevention and treatment of numb Chinese yam root rot, is by bacillus amyloliquefaciens CGMCC
No.12821 be inoculated in containing carbon source, nitrogen source and necessary nutrient without bacteria fermentation culture medium, carry out aerobic fementation and be made to contain
There is the liquid microbial microbial inoculum of above-mentioned viable bacteria.
Particular technique content in the present invention is also:
The fermentation medium is grouped as by the group of following mass percent:
Starch 0.3%-1.5%；Beancake powder 0.03%-0.1%；Calcium carbonate 0.05%-0.2%；Sucrose 0.08%-
0.5%；Ammonium sulfate 0.01%-0.08%；Dipotassium hydrogen phosphate trihydrate 0.08%-0.5%；Bitter salt 0.08%-
0.5%；Iron(III) chloride hexahydrate 0.008%-0.05%；Yeast extract 0.03%-0.15%；Potassium hydroxide 0.01%-0.1%；
GPE 0.005%-0.02%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
10 tons of fermenter volume, loading amount 60%-80%.It is inoculated with, fermented according to the inoculum concentration that volume ratio is 10%-15%
Temperature is 25-32 DEG C in journey, pressure 0.03-0.05MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 200-250M3/
Hr, 4-8 hours are 320-360M3/ hr, 8-12 hours are 420-470M3/ hr, 12-20 hours are 520-560M3/ hr, 20 hours
It is afterwards 600-630M3/hr；Fermentation is stirred after 4-8 hours, when the trophosome formation gemma for being not less than 95% in fermentation liquid and effectively
When viable bacteria is not less than 8,000,000,000/mL, as fermentation termination, it is packed and stored after quality inspection.
To shorten fermentation period, preferably and relatively conventional technical solution is, the bacillus amyloliquefaciens CGMCC
No.12821 is before accessing fermentation medium by expanding culture.
More preferably technical solution is that the expansion culture includes the preparation of test tube slant strain, Kolle flask inclined-plane
The preparation of strain and the preparation of seed liquor.
The preparation of the slant strains is will to be inoculated in sterile examination in bacillus amyloliquefaciens CGMCC No.12821
Test tube slant strain is made through culture in pipe slant strains culture medium；The culture medium of test tube slant strain by following quality raw material group
At:
5-15 grams of sucrose；0.2-1.0 grams of dipotassium hydrogen phosphate trihydrate；0.1-0.5 grams of bitter salt；Sodium chloride 0.1-
0.5 gram；0.5-3 grams of calcium carbonate；0.1-1.0 grams of yeast extract；15-22 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 25-32 DEG C, cultivation cycle 2-3 days.
The preparation of the Kolle flask slant strains is that test tube slant strain is inoculated in sterile Kolle flask slant strains
Kolle flask slant strains are made through culture in culture medium, and Kolle flask slant strains culture medium is made of the raw material of following quality:
0.5-2 grams of glucose；0.5-2 grams of peptone；0.5-2 grams of dipotassium hydrogen phosphate trihydrate；0.1-0.5 grams of ammonium sulfate；
0.3-1.5 grams of yeast extract；0.1-0.5 grams of bitter salt；15-22 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 60-80ml, culture temperature
Degree is 25-32 DEG C, cultivation cycle 2-3 days.
The preparation of the seed liquor is that Kolle flask slant strains are inoculated in sterile seed culture medium to be made through culture
Seed liquor, seed liquid culture medium are made of the raw material of following mass percentage:
Starch 0.2%-1.0%；Beancake powder 0.1%-0.7%；Sucrose 0.05%-0.3%；Calcium carbonate 0.05%-
0.3%；Ammonium sulfate 0.1%-1.0%；GPE 0.005%-0.015%；Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on 2-3 bottles of Kolle flask slant strains culture mediums under sterile water wash, by bacterium
Suspension is seeded in the seeding tank for being loaded with sterile seed culture medium.1 ton of seed tank volume, loading amount 60%-80%.Culture temperature
25-32 DEG C of degree, pressure 0.05MPa, 0-8 hour: ventilation quantity 50-70M3/ hr, after 8 hours: ventilation quantity 70-90M3/ hr,
Cultivation cycle 15-20 hours.
Culture terminal in the seed liquor preparation process is sampling microscopy observation upgrowth situation, when thallus is in logarithm
When growth period phase, bacteria containing amount reach technique require and without miscellaneous bacteria.
Speed of agitator is 730 revs/min behind described fermentation 4-8 hours.
The substantive distinguishing features and significant technological progress that the present invention has are:
1, the present invention has filled up the technological gap that there is no bacillus amyloliquefaciens for numb Chinese yam root rot prevention and treatment at present, passes through
Applicant detects the liquid microbial microbial inoculum using method preparation of the invention, and all technical meets GB20287-
2006 requirement.
2, the field comparison test that carries out through applicant in Baoding Dingxing County and Lixian County the result shows that, use present invention system
Standby liquid microbial microbial inoculum significantly improves numb Chinese yam root rot control efficiency than application fenaminosulf wettable powder, volume increase effect
Fruit significantly improves and numb Chinese yam stripe shape is straight, coarse, white color, abnormal rate are low.
Strain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC in the present invention
No.12821 system applicant separates acquirement from Hebei province, Dingxing County, Baoding fiber crops Chinese yam rhizosphere soil, and applicant is in 2016
Submit within August 8 days the China Committee for Culture Collection of Microorganisms for being located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 common
The preservation of microorganism center, depositary institution abbreviation CGMCC.
Specific embodiment
The invention will be further described with reference to embodiments, but not as a limitation of the invention, guarantor of the invention
Shield range is subject to the content of claim record, any equivalent technical elements replacement made according to specification, all without departing from
Protection scope of the present invention.
Embodiment 1
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.12821.
Application of the bacillus amyloliquefaciens in the prevention and treatment of numb Chinese yam root rot, is by bacillus amyloliquefaciens CGMCC
No.12821 be inoculated in containing carbon source, nitrogen source and necessary nutrient without bacteria fermentation culture medium, carry out aerobic fementation and be made to contain
There is the liquid microbial microbial inoculum of above-mentioned viable bacteria.
The fermentation medium is grouped as by the group of following mass percent:
Starch 1.5%；Beancake powder 0.1%；Calcium carbonate 0.2%；Sucrose 0.5%；Ammonium sulfate 0.08%；Three hypophosphite monohydrate hydrogen
Dipotassium 0.5%；Bitter salt 0.5%；Iron(III) chloride hexahydrate 0.05%；Yeast extract 0.15%；Potassium hydroxide 0.1%；
GPE 0.02%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
10 tons of fermenter volume, loading amount 80%.It is inoculated with according to the inoculum concentration that volume ratio is 15%, temperature is in fermentation process
32 DEG C, pressure 0.05MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 250M3/ hr, 4-8 hours are 360M3/ hr, 8-12
Hour is 470M3/ hr, 12-20 hours are 560M3/ hr is 630M after 20 hours3/hr；Fermentation is stirred after 4-8 hours, works as fermentation
When trophosome formation gemma and effective viable bacteria in liquid not less than 95% are not less than 8,000,000,000/mL, as fermentation termination, wrapped after quality inspection
It is packed into library.
The bacillus amyloliquefaciens CGMCC No.12821 is before accessing fermentation medium by expanding culture.
The expansion culture includes preparation, the preparation of Kolle flask slant strains and the system of seed liquor of test tube slant strain
It is standby.
The preparation of the slant strains is will to be inoculated in sterile examination in bacillus amyloliquefaciens CGMCC No.12821
Test tube slant strain is made through culture in pipe slant strains culture medium；The culture medium of test tube slant strain by following quality raw material group
At:
15 grams of sucrose；1.0 grams of dipotassium hydrogen phosphate trihydrate；0.5 gram of bitter salt；0.5 gram of sodium chloride；Calcium carbonate 3
Gram；1.0 grams of yeast extract；22 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 32 DEG C, cultivation cycle 2 days.
The preparation of the Kolle flask slant strains is that test tube slant strain is inoculated in sterile Kolle flask slant strains
Kolle flask slant strains are made through culture in culture medium, and Kolle flask slant strains culture medium is made of the raw material of following quality:
2 grams of glucose；2 grams of peptone；2 grams of dipotassium hydrogen phosphate trihydrate；0.5 gram of ammonium sulfate；1.5 grams of yeast extract；Seven water
Close 0.5 gram of magnesium sulfate；22 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 80ml, cultivation temperature
It is 32 DEG C, cultivation cycle 3 days.
The preparation of the seed liquor is that Kolle flask slant strains are inoculated in sterile seed culture medium to be made through culture
Seed liquor, seed liquid culture medium are made of the raw material of following mass percentage:
Starch 1.0%；Beancake powder 0.7%；Sucrose 0.3%；Calcium carbonate 0.3%；Ammonium sulfate 1.0%；GPE 0.015%；
Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on the lower 3 bottles of Kolle flasks slant strains culture medium of sterile water wash, bacterium is hanged
Liquid is seeded in the seeding tank for being loaded with sterile seed culture medium, and 1 ton of seed tank volume, loading amount 80%.32 DEG C of cultivation temperature,
Pressure 0.05MPa, 0-8 hour: ventilation quantity 70M3/ hr, after 8 hours: ventilation quantity 90M3/ hr, cultivation cycle 15 hours.
Culture terminal in the seed liquor preparation process is sampling microscopy observation upgrowth situation, when thallus is in logarithm
When growth period phase, bacteria containing amount reach technique require and without miscellaneous bacteria.
Speed of agitator is 730 revs/min behind described fermentation 4-8 hours.
Effective viable bacteria content is 81.2 hundred million/mL, no mould contamination, miscellaneous bacteria rate≤1%, pH in prepared microbial bacterial agent
It is 6.8.
Embodiment 2
The present embodiment the difference from embodiment 1 is that:
The fermentation medium is grouped as by the group of following mass percent:
Starch 0.3%；Beancake powder 0.03%；Calcium carbonate 0.05%；Sucrose 0.08%；Ammonium sulfate 0.01%；Three hydration phosphorus
Sour hydrogen dipotassium 0.08%；Bitter salt 0.08%；Iron(III) chloride hexahydrate 0.008%；Yeast extract 0.03%；Potassium hydroxide
0.01%；GPE 0.005%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
Fermentor be general-purpose type fermentor, 10 tons of volume, loading amount 60%.It is inoculated with according to the inoculum concentration that volume ratio is 10%,
Temperature is 25 DEG C in fermentation process, pressure 0.03MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 200M3/ hr, 4-8 are small
When be 320M3/ hr, 8-12 hours are 420M3/ hr, 12-20 hours are 520M3/ hr is 600M after 20 hours3/hr；Ferment 4-8
Start to stir after hour, when the trophosome for being not less than 95% in fermentation liquid forms gemma and effective viable bacteria is not less than 8,000,000,000/mL,
As fermentation termination, it is packed and stored after quality inspection.
The culture medium of test tube slant strain is made of the raw material of following quality:
5 grams of sucrose；0.2 gram of dipotassium hydrogen phosphate trihydrate；0.1 gram of bitter salt；0.1 gram of sodium chloride；Calcium carbonate 0.5
Gram；0.1 gram of yeast extract；15 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 25 DEG C, cultivation cycle 3 days.
Kolle flask slant strains culture medium is made of the raw material of following quality:
0.5 gram of glucose；0.5 gram of peptone；0.5 gram of dipotassium hydrogen phosphate trihydrate；0.1 gram of ammonium sulfate；Yeast extract 0.3
Gram；0.1 gram of bitter salt；15 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 60ml, cultivation temperature
It is 25 DEG C, cultivation cycle 3 days.
Seed liquid culture medium is made of the raw material of following mass percentage:
Starch 0.2%；Beancake powder 0.1%；Sucrose 0.05%；Calcium carbonate 0.05%；Ammonium sulfate 0.1%；GPE
0.005%；Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on the lower 2 bottles of Kolle flasks slant strains culture medium of sterile water wash, bacterium is hanged
Liquid is seeded in the seeding tank for being loaded with sterile seed culture medium, and 1 ton of seed tank volume, loading amount 60%.25 DEG C of cultivation temperature,
Pressure 0.05MPa, 0-8 hour: ventilation quantity 50M3/ hr, after 8 hours: ventilation quantity 70M3/ hr, cultivation cycle 20 hours.
Effective viable bacteria content is 81.5 hundred million/mL, no mould contamination, miscellaneous bacteria rate≤1%, pH in prepared microbial bacterial agent
It is 6.8.
Remaining content is the same as embodiment 1.
Embodiment 3
The present embodiment the difference from embodiment 1 is that:
The fermentation medium is grouped as by the group of following mass percent:
Starch 0.9%；Beancake powder 0.065%；Calcium carbonate 0.12%；Sucrose 0.29%；Ammonium sulfate 0.045%；Three hydrations
Dipotassium hydrogen phosphate 0.29%；Bitter salt 0.29%；Iron(III) chloride hexahydrate 0.029%；Yeast extract 0.09%；Hydroxide
Potassium 0.065%；GPE 0.012%-0.02%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
10 tons of fermenter volume, loading amount 75%.It is inoculated with according to the inoculum concentration that volume ratio is 13%, temperature is in fermentation process
29 DEG C, pressure 0.04MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 220M3/ hr, 4-8 hours are 340M3/ hr, 8-12
Hour is 450M3/ hr, 12-20 hours are 540M3/ hr is 615M after 20 hours3/hr；Fermentation is stirred after 4-8 hours, works as fermentation
When trophosome formation gemma and effective viable bacteria in liquid not less than 95% are not less than 8,000,000,000/mL, as fermentation termination, wrapped after quality inspection
It is packed into library.
The culture medium of test tube slant strain is made of the raw material of following quality:
10 grams of sucrose；0.6 gram of dipotassium hydrogen phosphate trihydrate；0.3 gram of bitter salt；0.3 gram of sodium chloride；Calcium carbonate
1.8 gram；0.55 gram of yeast extract；19 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 28 DEG C, cultivation cycle 2.5 days.
Kolle flask slant strains culture medium is made of the raw material of following quality:
1.25 grams of glucose；1.25 grams of peptone；1.25 grams of dipotassium hydrogen phosphate trihydrate；0.3 gram of ammonium sulfate；Yeast extract
0.9 gram；0.3 gram of bitter salt；18 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 70ml, cultivation temperature
It is 28 DEG C, cultivation cycle 2.5 days.
Seed liquid culture medium is made of the raw material of following mass percentage:
Starch 0.6%；Beancake powder 0.4%；Sucrose 0.17%；Calcium carbonate 0.17%；Ammonium sulfate 0.55%；GPE
0.01%；Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on the lower 2.5 bottles of Kolle flasks slant strains culture medium of sterile water wash, by bacterium
Suspension is seeded in the seeding tank for being loaded with sterile seed culture medium, and 1 ton of seed tank volume, loading amount 75%.Cultivation temperature 28
DEG C, pressure 0.03MPa, 0-8 hour: ventilation quantity 60M3/ hr, after 8 hours: ventilation quantity 80M3/ hr, cultivation cycle 18 are small
When.
Effective viable bacteria content is 82.5 hundred million/mL in prepared microbial bacterial agent.Without mould contamination, miscellaneous bacteria rate≤1%, pH
It is 6.9.
Remaining content is the same as embodiment 1.
Embodiment 4
The present embodiment the difference from embodiment 1 is that:
The fermentation medium is grouped as by the group of following mass percent:
Starch 0.6%；Beancake powder 0.04%；Calcium carbonate 0.08%；Sucrose 0.1%；Ammonium sulfate 0.02%；Three hypophosphite monohydrates
Hydrogen dipotassium 0.2%；Bitter salt 0.2%；Iron(III) chloride hexahydrate 0.02%；Yeast extract 0.05%；Potassium hydroxide
0.03%；GPE 0.008%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
10 tons of fermenter volume, loading amount 70%.It is inoculated with according to the inoculum concentration that volume ratio is 11%, temperature is in fermentation process
26 DEG C, pressure 0.035MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 210M3/ hr, 4-8 hours are 330M3/ hr, 8-
12 hours are 430M3/ hr, 12-20 hours are 530M3/ hr is 610M after 20 hours3/hr；Fermentation is stirred after 4-8 hours, works as hair
When trophosome formation gemma and effective viable bacteria in zymotic fluid not less than 95% are not less than 8,000,000,000/mL, as fermentation termination, after quality inspection
It is packed and stored.
The culture medium of test tube slant strain is made of the raw material of following quality:
7 grams of sucrose；0.4 gram of dipotassium hydrogen phosphate trihydrate；0.2 gram of bitter salt；0.2 gram of sodium chloride；Calcium carbonate 1
Gram；0.3 gram of yeast extract；16 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 26 DEG C, cultivation cycle 2.5 days.
Kolle flask slant strains culture medium is made of the raw material of following quality:
1 gram of glucose；1 gram of peptone；1 gram of dipotassium hydrogen phosphate trihydrate；0.2 gram of ammonium sulfate；0.6 gram of yeast extract；Six water
Close 0.2 gram of magnesium sulfate；16 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 65ml, cultivation temperature
It is 26 DEG C, cultivation cycle 2.5 days.
Seed liquid culture medium is made of the raw material of following mass percentage:
Starch 0.4%；Beancake powder 0.3%；Sucrose 0.1%；Calcium carbonate 0.1%；Ammonium sulfate 0.3%；GPE 0.007%；
Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on the lower 2 bottles of Kolle flasks slant strains culture medium of sterile water wash, bacterium is hanged
Liquid is seeded in the seeding tank for being loaded with sterile seed culture medium, and 1 ton of seed tank volume, loading amount 70%.26 DEG C of cultivation temperature,
Pressure 0.05MPa, 0-8 hour: ventilation quantity 55M3/ hr, after 8 hours: ventilation quantity 75M3/ hr, cultivation cycle 19 hours.
Effective viable bacteria content is 83.9 hundred million/mL in prepared microbial bacterial agent.Without mould contamination, miscellaneous bacteria rate≤1%, pH
It is 6.8.
Remaining content is the same as embodiment 1.
Embodiment 5
The present embodiment the difference from embodiment 1 is that:
The fermentation medium is grouped as by the group of following mass percent:
Starch 1.2%；Beancake powder 0.08%；Calcium carbonate 0.15%；Sucrose 0.4%；Ammonium sulfate 0.06%；Three hypophosphite monohydrates
Hydrogen dipotassium 0.4%；Bitter salt 0.4%；Iron(III) chloride hexahydrate 0.04%；Yeast extract 0.12%；Potassium hydroxide
0.08%；GPE 0.018%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
10 tons of fermenter volume, loading amount 65%.It is inoculated with according to the inoculum concentration that volume ratio is 12%, temperature is in fermentation process
30 DEG C, pressure 0.045MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 240M3/ hr, 4-8 hours are 350M3/ hr, 8-
12 hours are 460M3/ hr, 12-20 hours are 550M3/ hr is 620M after 20 hours3/hr；Fermentation is stirred after 4-8 hours, works as hair
When trophosome formation gemma and effective viable bacteria in zymotic fluid not less than 95% are not less than 8,000,000,000/mL, as fermentation termination, after quality inspection
It is packed and stored.
The culture medium of test tube slant strain is made of the raw material of following quality:
12 grams of sucrose；0.8 gram of dipotassium hydrogen phosphate trihydrate；0.4 gram of bitter salt；0.4 gram of sodium chloride；Calcium carbonate
2.5 gram；0.8 gram of yeast extract；21 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 30 DEG C, cultivation cycle 2 days.
Kolle flask slant strains culture medium is made of the raw material of following quality:
1.5 grams of glucose；1.5 grams of peptone；1.5 grams of dipotassium hydrogen phosphate trihydrate；0.4 gram of ammonium sulfate；Yeast extract 1.2
Gram；0.4 gram of bitter salt；21 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 75ml, cultivation temperature
It is 30 DEG C, cultivation cycle 2 days.
Seed liquid culture medium is made of the raw material of following mass percentage:
Starch 0.8%；Beancake powder 0.6%；Sucrose 0.2%；Calcium carbonate 0.2%；Ammonium sulfate 0.8%；GPE 0.012%；
Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on the lower 3 bottles of Kolle flasks slant strains culture medium of sterile water wash, bacterium is hanged
Liquid is seeded in the seeding tank for being loaded with sterile seed culture medium, and 1 ton of seed tank volume, loading amount 65%.31 DEG C of cultivation temperature,
Pressure 0.05MPa, 0-8 hour: ventilation quantity 65M3/ hr, after 8 hours: ventilation quantity 85M3/ hr, cultivation cycle 16 hours.
Effective viable bacteria content is 82.0 hundred million/mL in prepared microbial bacterial agent.Without mould contamination, miscellaneous bacteria rate≤1%, pH
It is 6.9.
Remaining content is the same as embodiment 1.
Show that microbial bacterial agent prepared by the method using 1-5 of the embodiment of the present invention meets through applicant's detection
The standard of GB20287-2006.
To verify effect of the invention, applicant carries out following field to the microbial bacterial agent prepared in embodiment 1-5 and answers
With test: Field information test is referring to " microbial manure field plot technique regulation and fertilizer efficiency evaluation guide (NY/T 1536-
2007) it executes.Test, which is divided to 2014 and 2,015 two, to be carried out.2014 experimental field in Baoding Dingxing County north Tian Cun, numb Chinese yam product
Kind is stick medicine, and sowing on April 29 harvested October 25；It is carried out in Baoding Lixian County with East Village within 2015, sowing on May 2,10
The moon 28 harvested.It is experimental field 2 years soils of continuous cropping, annual two pieces of selection is experimental field tested.Numb Chinese yam Pathogens of Root Rot
Bacterium is Rhizoctonia solani Kuhn.Fenaminosulf wettable powder is commercially available.
One, experimental design
Every group of test is all made of RANDOMIZED BLOCK DESIGN.Bacillus amyloliquefaciens microbial inoculum dosage is 15kg/hm2, fenaminosulf can
Wet powder dosage is 15kg/hm2If fenaminosulf wettable powder (T1), the bacillus amyloliquefaciens microbial inoculum (embodiment of the present invention
Bacillus amyloliquefaciens microbial inoculum, T prepared by 1-52-T6) and seven processing of clear water control (CK), 4 repetitions of every processing, cell
Area 60m2.Other management conditions such as each processing watering, fertilising are consistent.
Two, test method
Bacillus amyloliquefaciens microbial inoculum and fenaminosulf wettable powder are pressed into 450kg/hm respectively2It is diluted with water, with by spraying
Device, which is uniformly sprayed onto, to be broadcast in the numb Chinese yam potato seed planted, and earthing seals ditch immediately.
Three, pilot survey
(1) numb Chinese yam morbidity survey: 30d starts numb Chinese yam root rot initial inquiry after numb Chinese yam field planting emergence, per small
Area investigates the absorption root of 30 plants of numb Chinese yams, investigates 1 time every 15d.Relative control effect is calculated before harvest.
Disease index=[Σ (the old complaint radicals at different levels × disease grade value)/(investigating total root radical × highest disease grade value)] ×
100
Relative control effect (%)=[(control disease index-processing disease index)/control disease index] × 100
(2) numb Chinese yam yield investigation: each processing harvests respectively, and each processing takes 30 plants at random, is investigated, and block is claimed
Gross weight calculates yield.
Four, test result such as table 1.
The control efficiency of the numb Chinese yam root rot of table 1 and influence to yield
Following result seen from table 1:
1, to the preventive effect of numb Chinese yam root rot
Bacillus amyloliquefaciens preparation relative control effect prepared by the method for embodiment 1-5 reaches in the application present invention
89.6% or more.Respectively higher than apply the T of chemical agent fenaminosulf wettable powder1Handle 4.69%-9.58%.It can be seen that use
The bacillus amyloliquefaciens microbial inoculum of method preparation of the invention has very high preventive effect to numb Chinese yam root rot.
2, to the effect of increasing production of numb Chinese yam
In terms of yield result, the control treatment on four pieces of ground seriously affects numb Chinese yam yield, significantly since disease incidence is higher
Lower than T1-T6Processing, and T2-T6Processing yield compares T respectively1High 7.48%-11.7% is handled, difference is significant.It can be seen that the present invention
The bacillus amyloliquefaciens microbial inoculum of method preparation is also significant to the effect of increasing production of numb Chinese yam.From the straight and neat of numb Chinese yam finished product
From the point of view of degree, the numb Chinese yam stripe shape of bacillus amyloliquefaciens microbial inoculum prepared by method of the application using 1-5 of the embodiment of the present invention is suitable
Directly, coarse, white color, abnormal rate is low.
Claims (7)
1. a bacillus amyloliquefaciens, classification naming is bacillus amyloliquefaciens Bacillus amyloli quefaciens,
Deposit number is CGMCC No.12821.
2. application of the bacillus amyloliquefaciens in the prevention and treatment of numb Chinese yam root rot, it is characterised in that being will be as described in claim 1
Bacillus amyloliquefaciens be inoculated in containing carbon source, nitrogen source and necessary nutrient without bacteria fermentation culture medium, carry out ventilation hair
The liquid microbial microbial inoculum containing viable bacteria is made in ferment；
The fermentation medium is grouped as by the group of following mass percent:
Starch 0.3%-1.5%；Beancake powder 0.03%-0.1%；Calcium carbonate 0.05%-0.2%；Sucrose 0.08%-0.5%；Sulphur
Sour ammonium 0.01%-0.08%；Dipotassium hydrogen phosphate trihydrate 0.08%-0.5%；Bitter salt 0.08%-0.5%；Six water
Close ferric trichloride 0.008%-0.05%；Yeast extract 0.03%-0.15%；Potassium hydroxide 0.01%-0.1%；GPE
0.005%-0.02%；Surplus is tap water；PH=7.2-7.4；
The process conditions of aerobic fementation are as follows:
It 10 tons of fermenter volume, loading amount 60%-80%, is inoculated with according to the inoculum concentration that volume ratio is 10%-15%, in fermentation process
Temperature is 25 DEG C -32 DEG C, pressure 0.03MPa-0.05MPa；Ventilation quantity in fermentation process are as follows: 0-4 hours are 200M3/hr-
250M3/ hr, 4-8 hours are 320M3/hr-360M3/ hr, 8-12 hours are 420M3/hr-470M3/ hr, 12-20 hour are
520M3/hr-560M3/ hr is 600M after 20 hours3/hr-630M3/hr；Fermentation is stirred after 4-8 hours, when not low in fermentation liquid
When 95% trophosome forms gemma and effective viable bacteria is not less than 8,000,000,000/mL, as fermentation termination；
For the bacillus amyloliquefaciens by expanding culture before accessing fermentation medium, expanding culture includes test tube slant bacterium
Preparation, the preparation of Kolle flask slant strains and the preparation of seed liquor of kind.
3. application of the bacillus amyloliquefaciens according to claim 2 in the prevention and treatment of numb Chinese yam root rot, it is characterised in that
The preparation of the slant strains is that sterile test tube slant bacterium will be inoculated in bacillus amyloliquefaciens CGMCC No.12821
Test tube slant strain is made through culture in kind culture medium；The culture medium of test tube slant strain is made of the raw material of following quality:
5-15 grams of sucrose；0.2-1.0 grams of dipotassium hydrogen phosphate trihydrate；0.1-0.5 grams of bitter salt；Sodium chloride 0.1-0.5
Gram；0.5-3 grams of calcium carbonate；0.1-1.0 grams of yeast extract；15-22 grams of agar；1000 milliliters of water；PH=7.2；
The condition of culture of test tube slant strain is:
Temperature is 25-32 DEG C, cultivation cycle 2-3 days.
4. application of the bacillus amyloliquefaciens according to claim 2 in the prevention and treatment of numb Chinese yam root rot, it is characterised in that
The preparation of the Kolle flask slant strains is that test tube slant strain is inoculated in sterile Kolle flask slant strains culture medium warp
Kolle flask slant strains are made in culture, and Kolle flask slant strains culture medium is made of the raw material of following quality:
0.5-2 grams of glucose；0.5-2 grams of peptone；0.5-2 grams of dipotassium hydrogen phosphate trihydrate；0.1-0.5 grams of ammonium sulfate；Yeast
0.3-1.5 grams of cream；0.1-0.5 grams of bitter salt；15-22 grams of agar；Water 1000ml；PH=7.2；
The condition of culture of Kolle flask slant strains is:
Above-mentioned Kolle flask slant strains culture medium is fitted into 250ml Kolle flask according to the amount of every bottle of 60-80ml, cultivation temperature is
25-32 DEG C, cultivation cycle 2-3 days.
5. application of the bacillus amyloliquefaciens according to claim 2 in the prevention and treatment of numb Chinese yam root rot, it is characterised in that
The preparation of the seed liquor is Kolle flask slant strains to be inoculated in sterile seed culture medium seed liquor is made through culture, is planted
Sub- liquid culture medium is made of the raw material of following mass percentage:
Starch 0.2%-1.0%；Beancake powder 0.1%-0.7%；Sucrose 0.05%-0.3%；Calcium carbonate 0.05%-0.3%；Sulphur
Sour ammonium 0.1%-1.0%；GPE 0.005%-0.015%；Surplus is tap water；PH=7.2；
The condition of culture of seed liquor is:
Bacteria suspension is made with the strain grown on 2-3 bottles of Kolle flask slant strains culture mediums under sterile water wash, by bacteria suspension
It is seeded in the seeding tank for being loaded with sterile seed culture medium, 1 ton of seed tank volume, loading amount 60%-80%, cultivation temperature
25-32 DEG C, pressure 0.05MPa, 0-8 hour: ventilation quantity 50-70M3/ hr, after 8 hours: ventilation quantity 70-90M3/ hr, training
It supports period 15-20 hours.
6. application of the bacillus amyloliquefaciens according to claim 5 in the prevention and treatment of numb Chinese yam root rot, it is characterised in that
Culture terminal in the seed liquor preparation process is sampling microscopy observation upgrowth situation, when thallus is in logarithmic phase growth period
When, bacteria containing amount reach technique require and without miscellaneous bacteria.
7. application of the bacillus amyloliquefaciens according to claim 2 in the prevention and treatment of numb Chinese yam root rot, it is characterised in that
Speed of agitator is 730 revs/min behind described fermentation 4-8 hours.
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