CN105191988B - A kind of bacillus amyloliquefaciens preparation and its application - Google Patents

A kind of bacillus amyloliquefaciens preparation and its application Download PDF

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CN105191988B
CN105191988B CN201510702267.XA CN201510702267A CN105191988B CN 105191988 B CN105191988 B CN 105191988B CN 201510702267 A CN201510702267 A CN 201510702267A CN 105191988 B CN105191988 B CN 105191988B
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bacillus amyloliquefaciens
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corn
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bacillusa
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CN105191988A (en
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刘洪亮
王慧
于琳
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刘洪亮
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Abstract

A kind of bacillus amyloliquefaciens preparation and its application, the present invention relates to a kind of bacillus amyloliquefaciens preparation and its application.The invention aims to solve it is existing prevent and treat chemical agent environmental pollution in the leaf blight of corn, dangerous to people and animals, germ is also easy to produce the resistance to the action of a drug, and the problem of to corn yield increasing DeGrain.The present invention's includes 0.01~0.02% zinc sulfate, bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1 01 of 0.01~0.02% boric acid, 0.1~0.2% polysorbas20 and surplus by mass fraction.The application of the present invention refers to the application on increase corn yield while Exserohilum turcicum is suppressed.The present invention is 80.8% to the preventive effect of the leaf blight of corn, and corn yield increasing rate is 18.26%.The present invention is applied to agriculture field.

Description

A kind of bacillus amyloliquefaciens preparation and its application
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens preparation and its application.
Background technology
The leaf blight of corn is also known as cecospora spot, coal line disease, dead leaf disease, leaf spot etc..The blade of main corn of causing harm, leaf sheath and Bract.Blade, which is caught an illness first, there is water stain shape cinerous spot, is then extended along vein to two ends, forms edge crineous, center Filbert or caesious big spot.The normal lobe of later stage scab.Scab is merged when serious, and blade turns yellow withered.When moist on scab There is the mould layer of a large amount of grey blacks.Lower blade is first fallen ill.Chlorisis scab is shown as on monogenic disease-resistant variety, scab is smaller, Parallel with vein, color and luster is yellowish green or filbert, surrounding crineous, and some show as necrotic plaque.
The prevention and controls of the leaf blight of corn mainly have plantation disease-resistant variety, strengthen cultural control and chemical control.Medicament is prevented Control aspect main based on chemical agent, and there is pollution environment in chemical agent, the lasting period is short, and pathogen easily develops immunity to drugs The problems such as, and existing bacteria agent is just for preventing and treating pathogen, it is impossible to play a part of corn yield increasing.
The content of the invention
The invention aims to solve it is existing prevent and treat chemical agent environmental pollution in the leaf blight of corn, it is uneasy to people and animals Entirely, germ is also easy to produce the resistance to the action of a drug, and there is provided a kind of bacillus amyloliquefaciens preparation the problem of to corn yield increasing DeGrain And its application.
1st, a kind of bacillus amyloliquefaciens preparation of the invention by mass fraction include 0.01~0.02% zinc sulfate, 0.01~ The bacillus amyloliquefaciens (Bacillusa myloliquefaciens) of 0.02% boric acid, 0.1~0.2% polysorbas20 and surplus KB1-01 zymotic fluids, wherein preparing bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 zymotic fluids The constituent content that every 1 liter of fermentation medium is:Glucose 0.1%, analysis for soybean powder 0.4%, peptone 0.1%, yeast extract 0.05%, The water of potassium dihydrogen phosphate 0.12%, magnesium sulfate 0.024% and surplus;Fermentation condition is:32 DEG C of temperature, pH7.0~7.2, rotating speed 170r/min, incubation time 48h, inoculum concentration 2%.
A kind of application of bacillus amyloliquefaciens preparation of the present invention refers to increase while Exserohilum turcicum is suppressed Application on corn yield.
The present invention is prepared into efficient, wide with bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 Spectrum, nontoxic, free of contamination environmental Biocontrol microorganism microbial inoculum, the microbial inoculum are environmentally friendly, pathogen will not be made to develop immunity to drugs, And determined by field efficacy, it is 80.8% to the preventive effect of the leaf blight of corn to take out 40d after hero, is significantly higher than medicament (carbendazim) right , in addition can be quickly numerous by bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 floras according to 60.1% Grow and capture site, secrete a variety of antibiotics, multiple diseases can be prevented;Application effect of the microbial inoculum on corn show, Under the conditions of conventional agrotechnical measure, corn, which is sprayed, can be obviously promoted carrying for plant growth and fringe yield after microbial bacterial agent Height, reduces prominent sharp rate, spike length, row grain number and 100-grain weight increase 1.4cm, 1.8,2.4g respectively compared with spraying blank control, beautiful Rice rate of growth is 18.26%.The microbial inoculum 14d, second of 14d are being used, 20d after hero is being taken out, 40d after hero is taken out, to the leaf blight of corn Prevention effect it is good, illustrate that the microbial inoculum lasting period is long.
Embodiment
Embodiment one:A kind of bacillus amyloliquefaciens preparation of present embodiment by mass fraction comprising 0.01~ 0.02% zinc sulfate, the bacillus amyloliquefaciens of 0.01~0.02% boric acid, 0.1~0.2% polysorbas20 and surplus (Bacillusa myloliquefaciens) KB1-01 zymotic fluids, wherein preparing bacillus amyloliquefaciens (Bacillusamyloliquefaciens) constituent content of every 1 liter of the fermentation medium of KB1-01 zymotic fluids is:Glucose 0.1%th, analysis for soybean powder 0.4%, peptone 0.1%, yeast extract 0.05%, potassium dihydrogen phosphate 0.12%, magnesium sulfate 0.024% and remaining The water of amount;Fermentation condition is:32 DEG C of temperature, pH7.0~7.2, rotating speed 170r/min, incubation time 48h, inoculum concentration 2%.
The present invention is prepared into efficient, wide with bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 Spectrum, nontoxic, free of contamination environmental Biocontrol microorganism microbial inoculum, the microbial inoculum are environmentally friendly, pathogen will not be made to develop immunity to drugs.
Embodiment two:Present embodiment is pressed from bacillus amyloliquefaciens preparation unlike embodiment one Mass fraction includes 0.02% zinc sulfate, 0.02% boric acid, 0.1% polysorbas20 and 99.86% bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 zymotic fluids, other are identical with embodiment one.
Embodiment three:Present embodiment from unlike embodiment one or two:Described solution starch bud It is general that spore bacillus (Bacillusa myloliquefaciens) KB1-01 is preserved in China Committee for Culture Collection of Microorganisms Logical microorganism center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is on 09 12nd, 2014, Deposit number is CGMCC No.9649, and other are identical with embodiment one or two.
Present embodiment bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 screening technique For:From the land-reclaimable Harbin in Heilongjiang Province, red prosperity, Mudanjiang, 93, Suihua, Bei'an, seven, Bao Quan ridges branch office's collection corn root 15 parts of border pedotheque.
By from the soil sample natural air drying of collection.Every part of soil sample weighs 10g, is put into and fills the three of 90ml sterilized waters and bead In the bottle of angle, acutely vibrated on shaking table and be coated with PDA plate after carrying out gradient dilution with sterilized water after 30min, in 25 DEG C of incubators Interior culture 24h or so, the obvious single bacterium colony of picking colony morphological differences enters primary dcreening operation.
Using Exserohilum turcicum as target bacterium, separated bacterium is screened using flat board face-off method.Will be for examination disease fungus Bacteria cake (7mm) moves to flat board center, at the 25mm of bacterium both sides, streak inoculation Antagonistic Fungi, using only connect pathogen as pair According to often handling 3 wares.25 DEG C of incubator cultures 4~6 days are placed in, 1182 plants of bacterial strains for having inhibitory action to target bacteria growing are selected Into secondary screening.Each 4 repetitions of bacterial strain, same method culture simultaneously measures inhibition zone radius (bacterial clump center to pathogen Mycelia edge) size, filter out that inhibition zone radius is larger, by suppression cause of disease mycelia edge is concordant and the lasting bacterium of antagonism Strain, by secondary screening, obtains bacterial strain KB1-01, and its average antibacterial band is 12.8mm, and bacteriostasis rate reaches 81.91%.
After flat board face-off growth method culture, bacterial strain KB1-01 is to rice blast fungus, Rhizoctonia solani Kuhn, Ma Ling Potato early epidemic germ, tomato wilt bacterium, Fusarium graminearum Fusarium oxysporum and rod method inhibitory action as shown in table 1, by table 1 Understand, bacterial strain KB1-01 shows the antagonism of wide spectrum to different disease funguses, but bacterial strain KB1-01 is to various pathogenic bacteria The antagonism intensity shown difference, this is probably to be made by the mycelium construction of different disease funguses etc. is different Into.
Table 1
Screen obtained bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 cellular morphology It is as shown in table 2 with physical and chemical experiment result:
Table 2
Molecule is carried out to bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 that screening is obtained Identification, is carried out by the following method:
Bacterial strain KB1-01 DNA is extracted using CTAB methods;KB1-01 DNA is expanded and surveyed using universal primer It is fixed;PCR primer is sequenced after purification, through sequencing, and sample 16S rDNA clip sizes to be identified are 1382bp.By this sequence Sequence homology is carried out with the data in ncbi database to compare, and finds the bacterial strain and bacillus amyloliquefaciens (Bacillusa Myloliquefaciens affiliation) recently, and is finally named as bacillus amyloliquefaciens (Bacillusamyloliquefaciens)KB1-01。
Embodiment four:Unlike one of present embodiment and embodiment one to three:Described Xie Dian You effects Jun Shuo living≤500,000,000/milliliter, gemma in afnyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 zymotic fluids Shuai≤90%, other are identical with one of embodiment one to three.
Embodiment five:A kind of application of bacillus amyloliquefaciens preparation of present embodiment refers to big in suppression corn Increase the application on corn yield while pinta bacterium.
Application effect of the bacillus amyloliquefaciens preparation of present embodiment on corn shows, in conventional agrotechnical measure bar Under part, corn, which is sprayed, can be obviously promoted plant growth and the raising of fringe yield after microbial bacterial agent, reduce prominent sharp rate, with Spray blank control increases 1.4cm, 1.8,2.4g respectively compared to spike length, row grain number and 100-grain weight, and corn yield increasing rate is 18.26%.The microbial inoculum 14d, second of 14d are being used, 20d after hero is being taken out, takes out 40d after hero, to the prevention effect of the leaf blight of corn Well, illustrate that the microbial inoculum lasting period is long.
Embodiment six:Present embodiment from unlike embodiment five:Said preparation and water are in mass ratio 1:Used after 500 ratio mixing, other are identical with embodiment five.
Beneficial effects of the present invention are verified by tests below:
Experiment 1, a kind of bacillus amyloliquefaciens preparation of this experiment include 0.02% zinc sulfate, 0.02% boron by mass fraction Acid, 0.1% polysorbas20 and 99.86 bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 fermentations Liquid, wherein preparing the fermentation medium of bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 zymotic fluids Every 1 liter of constituent content is:Glucose 0.1%, analysis for soybean powder 0.4%, peptone 0.1%, yeast extract 0.05%, potassium dihydrogen phosphate 0.12%, the water of magnesium sulfate 0.024% and surplus;Fermentation condition is:32 DEG C of temperature, pH7.0~7.2, rotating speed 170r/min, training Support time 48h, inoculum concentration 2%.
Bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 in this experiment is preserved in China Microbiological Culture Collection administration committee common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Number, preservation date is on 09 12nd, 2014, and deposit number is CGMCC No.9649.
In this experiment in bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 zymotic fluids effectively Jun Shuo living≤500,000,000/milliliter, gemma Shuai≤90%.
Bacteria agent is determined by the field control effectiveness tests of 2 years and prevents and treats leaf blight of corn application dosage, determines its preventing and treating effect Fruit and the influence to corn yield.Different disposal is observed on emergence of corn and seedling-growing time either with or without influence;Investigate the hair of corn Sick rate, the experimental plot such as growth traits, growing way of the seedlings investigation respectively between processing is corn continuous cropping plot for many years.Dispenser period is corn The leaf blight early stage 7-8 leaf phases, 10-14d is spaced, carries out second of medication, 1.5-1.75kg/ hectares of dosage.
Using RANDOMIZED BLOCK DESIGN, experiment uses great Qu methods of comparison, and secondary repetition, 1.1 meters of row spacing, row is long 15 meters, 5 ridges Area, per 165 square metres of processing area, the maturity period, which is often handled, takes middle two rows even after taking 5 square metres, carries out species test, paid survey production.
Experimental field belong to meadow chernozemic soil, Application Ratio:For N:P:K=20:17:6,4kg/ mus of seed manure Diammonium phosphate (DAP), deep fertilizer spraying 10.48kg/ mus of Diammonium phosphate (DAP), 4.6kg/ mus of urea, 6kg/ mus of potassium sulfate topdresses:10 kgs/acre of urea, tests kind dolantin Sub- No. 2, the design land measure equal to fifteen mu in most parts of the Northeast is kept a full stand of seedings 90,000 plants, field management:Subsoiling once, intertillage once, chemical weed control, Maize at Seedling Stage is with 550/ liter of nitre Milliliter/mu of sulphur atrazine suspending agent 133, is watered 10 kgs/acre of cauline leaf process.
The compound method of KB1-01 500 times of liquid is bacillus amyloliquefaciens agent and water in mass ratio 1:500 ratio is mixed Close;KB1-01 1000 times of liquid making methods are bacillus amyloliquefaciens agent and water in mass ratio 1:1000 ratio mixing; KB1-01 2000 times of liquid making methods are bacillus amyloliquefaciens agent and water in mass ratio 1:2000 ratio mixing.
Field reagent result of the test shows (table 3) within 2014:By being determined to biological agent various dose field efficacy, place 1 pair of best preventive effect of leaf blight of corn prevention effect of reason is 80.8%, is significantly higher than medicament (carbendazim) control 60.1%.Volume increase effect Fruit is 18.26%.
Table 3
Cross above-mentioned experiment and understand that bacillus amyloliquefaciens (Bacillusa myloliquefaciens) KB1-01 is to environment Close friend, the lasting period is long, pathogen will not be made to develop immunity to drugs, and is determined by field efficacy, and the preventive effect to the leaf blight of corn is 80.8%, it is significantly higher than medicament (carbendazim) control 60.1%, corn yield increasing rate is 18.26%.In addition can be by solving starch bud Quickly site is captured in breeding to spore bacillus (Bacillusa myloliquefaciens) KB1-01 floras, secretes a variety of antibionts Matter, can prevent multiple diseases;It is efficient, wide spectrum, nontoxic, free of contamination environmental Biocontrol microorganism preparation.

Claims (5)

1. a kind of bacillus amyloliquefaciens preparation, it is characterised in that it by mass fraction be by 0.01~0.02% zinc sulfate, The bacillus amyloliquefaciens KB1-01 zymotic fluid groups of 0.01~0.02% boric acid, 0.1~0.2% polysorbas20 and surplus into, wherein The constituent content for preparing every 1 liter of the fermentation medium of bacillus amyloliquefaciens KB1-01 zymotic fluids is:Glucose 0.1%, soya bean Powder 0.4%, peptone 0.1%, yeast extract 0.05%, potassium dihydrogen phosphate 0.12%, the water of magnesium sulfate 0.024% and surplus;Hair Ferment condition is:32 DEG C of temperature, pH7.0~7.2, rotating speed 170r/min, incubation time 48h, inoculum concentration 2%;Described solution starch Bacillus KB1-01 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is Beijing The institute 3 of city Chaoyang District North Star West Road 1, preservation date is on 09 12nd, 2014, and deposit number is CGMCC No.9649.
2. a kind of bacillus amyloliquefaciens preparation according to claim 1, it is characterised in that bacillus amyloliquefaciens preparation It is by 0.02% zinc sulfate, 0.02% boric acid, 0.1% polysorbas20 and 99.86% bacillus amyloliquefaciens by mass fraction KB1-01 zymotic fluid groups into.
3. a kind of bacillus amyloliquefaciens preparation according to claim 1, it is characterised in that described solution starch gemma bar You effects Jun Shuo living≤500,000,000/milliliter, gemma Shuai≤90% in bacterium KB1-01 zymotic fluids.
4. the application of bacillus amyloliquefaciens preparation as claimed in claim 1, it is characterised in that the microbial inoculum is big in suppression corn Increase the application on corn yield while pinta bacterium.
5. the application of a kind of bacillus amyloliquefaciens preparation according to claim 4, it is characterised in that said preparation is pressed with water Mass ratio 1:Used after 500 ratio mixing.
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CN102864100A (en) * 2012-08-16 2013-01-09 北京雷力农用化学有限公司 Bacillus amyloliquefaciens and application thereof
CN103215209A (en) * 2013-04-24 2013-07-24 牛赡光 Bacillus amyloliquefaciens and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101423812A (en) * 2008-12-17 2009-05-06 河南省农业科学院 Bacillus amyloliquefaciens and microbiological preparation and preparation method thereof
CN102864100A (en) * 2012-08-16 2013-01-09 北京雷力农用化学有限公司 Bacillus amyloliquefaciens and application thereof
CN103215209A (en) * 2013-04-24 2013-07-24 牛赡光 Bacillus amyloliquefaciens and application thereof

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