CN106367367A - Biocontrol compound microbial liquid, compound microbial fertilizer and production method - Google Patents
Biocontrol compound microbial liquid, compound microbial fertilizer and production method Download PDFInfo
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- CN106367367A CN106367367A CN201610756759.1A CN201610756759A CN106367367A CN 106367367 A CN106367367 A CN 106367367A CN 201610756759 A CN201610756759 A CN 201610756759A CN 106367367 A CN106367367 A CN 106367367A
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- fertilizer
- bacterium solution
- biocontrol
- bacillus
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- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical group [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
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- 235000010339 sodium tetraborate Nutrition 0.000 description 1
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- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
- C05F5/008—Waste from biochemical processing of material, e.g. fermentation, breweries
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Botany (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental & Geological Engineering (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Pest Control & Pesticides (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses biocontrol compound microbial liquid, wherein the biocontrol compound microbial liquid includes bacillus subtilis LVLE14 bacterial liquid and bacillus amylolique faciens LVLE15 bacterial liquid. Meanwhile, the invention provides a liquid compound microbial fertilizer and a solid compound microbial fertilizer produced from the compound microbial liquid, and the invention provides a production method of the liquid compound microbial fertilizer and a production method of the solid compound microbial fertilizer. The biocontrol compound fertilizer provided by the invention has an outstanding control effect on anthracnose; the biocontrol compound fertilizer, after being applied, has both functions of disease preventing and fertilizer efficiency; the biocontrol compound fertilizer can improve disease resistance of crops as well as absorption rate and utilization rate of the fertilizer, and the biocontrol compound fertilizer can keep vigorous growth of the crops; the biocontrol compound fertilizer, in turn, can also promote the reproduction of biocontrol bacteria and can improve the disease resistance of the crops; and the biocontrol compound fertilizer is conducive to the formation of a good agricultural ecological system.
Description
Technical field
The present invention relates to one kind can prevent and kill off various crop disease complex microorganism bacterium solution and in particular to this biocontrol water bacterium
Liquid, bio-feritlizer and production method.
Background technology
China's controlling disease substantially adopts chemical pesticide at present, and chemical pesticide control is with its strong toxicity, prevention effect
Good, low cost and generally welcome by peasant.The annual pesticide producing of China kind, process preparation kind more than 500 about more than 250, active compound is given birth to
Produce more than 80 ten thousand tons, occupy the 2nd, the world.Pesticide annual consumption is ten thousand tons of 80-100, occupies first place in the world.But in a large number using chemical agriculture
Medicine, one is to cause pollution by pesticides to environment, and two is to bring Practice for Pesticide Residue in Agricultural Products problem, causes prestige to human health
The side of body.For reducing the use of chemical pesticide, cultural control in recent years, biological green preventing and treating are at home and abroad progressively risen.
Exploitation biological pesticide and there is the microbial-bacterial fertilizer of biological and ecological methods to prevent plant disease, pests, and erosion characteristic have become as a kind of international trend.The world is each
State just announced in 90 years to cancel 91 kinds of chemical pesticides in the use reducing chemical pesticide that takes practical steps, such as U.S. epa
Registration.The development and application of biological pesticide is widely paid close attention to and is paid attention to, and has become pesticide career field in the world at present new
Research and development focus.Biological control with regard to soil-borne disease begins one's study the external twenties, and such as nineteen twenty-one c.hartley report is permissible
Prevent and treat the mould root rot of corruption of seedling with ten several common soil earth saprophytic fungus and antibacterial.40th, the fifties develops Antagonism and puts
Line bacterium and produced antibiotic, turn to and find other antagonistic microbes and biological and ecological methods to prevent plant disease, pests, and erosion technical research the sixties.Report in the world
The bio-control factors being applied to soil-borne disease are a lot, including funguses, antibacterial, viral phage and defence line bacterium etc., wherein portioned product
For merchandized handling, and play more and more important effect in integrated pest prevention and control and agricultural sustainable development.Mesh
Before, the output value of biological pesticide is annual in the world is all increasing in the speed of 10-20%.Reduce the use of chemical synthetic pesticide, send out
Exhibition biological pesticide become a kind of trend and direction it is contemplated that.
Based on agricultural antibiotic, mainstream product includes jinggangmycin, blasticidin-Ss, multi-resistance for the exploitation of domestic biological pesticide
Mycin, kasugarnycin, valida and mildiomycin etc. are used for preventing and treating fungal disease, and streptomycin, penicillin and oxytetracycline etc. are used
To prevent and treat bacteriosises；The biological living pesticide succeeded in developing at present also has sub- treasured, power treasured, waxy Bacillus, yield increasing fungus (to plant
Thing growth regulator), k84, e26, Pseudomonas alba and the fluorescence vacation bacteria preparation such as monospore, for the preventing and treating of soil-borne disease.Its
The anti-crown gall microbial inoculum k84 registered registration at present of middle China Agricultural University exploitation, product is 2,000,000 cfu/ milliliter radiation soil bars
Bacterium wet-milling, reaches 100% to root knot effects such as kernel approaches (Fructus Persicae, Lee, Fructus Pruni pseudocerasi, Fructus Pruni etc.)；To pomaceous fruit and forest, flowers root knot
There are better effects.The gemma bacillus agent also registered registration at present of Yunnan agricultural university exploitation, product is 1,000,000,000/g bacillus cereuss wp
(hundred resist), there is to rice sheath blight disease good preventive effect.It is (especially no public that other parts scientific achievement has also been applied to production practices
Evil food production base), and achieve good ecology, Social and economic benef@.
Content of the invention
Therefore, the technical problem to be solved in the present invention is to provide a kind of composite microbial bacteria with good biocontrol effect
Liquid, especially has the bacterial strain of good biocontrol effect to cucumber disease.The invention solves the problems that another technical problem, be profit
Produce bio-feritlizer with this bacterial strain, quality meets the requirement of the Ministry of Agriculture " composite microbiological fertilizer " (ny/t798-2015).This
The bright production method that this fertilizer is also provided.
The technical scheme is that, a kind of biocontrol water complex microorganism bacterium solution, described biocontrol water complex microorganism bacterium solution
Including bacillus subtilises (bacillus subtilis) lvle14 bacterium solution and bacillus amyloliquefaciens (bacillus
Amylolique faciens) lvle15 bacterium solution.Both bacterium solution are bacillus subtilises (bacillus subtilis) respectively
Lvle14 and bacillus amyloliquefaciens (bacillus amylolique faciens) fermenting and producing obtain.
Bacillus subtilises (bacillus subtilis) lvle14, is deposited in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation date is in June, 2016
20, deposit number was cgmcc no.12639.
Bacillus amyloliquefaciens (bacillus amylolique faciens) lvle15, is deposited in China Microbiological bacterium
Plant preservation administration committee common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation day
Phase is on June 20th, 2016, and deposit number is cgmcc no.12640.
Preferably, bacillus subtilises lvle14 and bacillus amyloliquefaciens lvle15 in described complex microorganism bacterium solution
Mass ratio be 1-4:1.It is further preferred that bacillus subtilises lvle14 conciliates starch spore in described complex microorganism bacterium solution
The mass ratio of bacillus lvle15 is 2-3:1.
Biocontrol water complex microorganism bacterium solution according to the present invention is it is preferred that described bacillus subtilises lvle14 bacterium solution
The fermentation condition of fermenting and producing is: ph6.5-7.5, and cultivation temperature is 20-40 DEG C, initial rotating speed 100-200rpm, and ventilation is
0.5-1vvm, cultivates 35-40 hour, and fermentation middle and late stage row becomes spore, spore rate up to more than 95%, the effective work in fermentation liquid
Bacterium number is in 10,000,000,000/more than ml；
The fermentation condition of described bacillus amyloliquefaciens lvle15 bacterium solution fermenting and producing is: ph is 6.5-8.0, cultivation temperature
For 30-45 DEG C, initial rotating speed 120-200rpm, ventilation is 0.5-1vvm, cultivates 35-45 hour, and fermentation middle and late stage row becomes bud
Spore, spore rate up to more than 93%, the living bacteria count in fermentation liquid is in 15,000,000,000/more than ml.
I.e. the living bacteria count of above-mentioned bacillus subtilises (bacillus subtilis) lvle14 bacterium solution 10,000,000,000/
The living bacteria count of more than ml, bacillus amyloliquefaciens (bacillus amylolique faciens) lvle15 is 15,000,000,000
Individual/more than ml.
Bacillus subtilises (bacillus subtilis) lvle14 characteristic: spore 0.6-0.9 × 1.0-1.5 micron, ellipse
Circle arrives column, no pod membrane, and after sporulation, thalline does not expand.Bacterium colony rough surface is opaque, Gram-positive, optimal temperature
30-40 DEG C, ph6.5-7.5.
Bacillus amyloliquefaciens (bacillus amylolique faciens) lvle15 characteristic: thalline is shaft-like, size
0.3-2.2 × 1.2-7.0 micron.Gram-positive, optimum growth temperature 25-35 DEG C, ph is neutral.
Preferably, bacillus subtilises (bacillus subtilis) lvle14 strain fermentation culture medium prescription: solvable
Property starch 20-40g, analysis for soybean powder 5-30g, sucrose 1-10g, peptone 2-8g, bean cake 1-6g, k2hpo41-5g、mgso40.2-
3g, bubble enemy 0.1-2g, moisturizing is to 1000 milliliters.
Bacillus amyloliquefaciens (bacillus amylolique faciens) lvle15 strain fermentation culture medium prescription is
Semen Maydis powder 20-30g, soybean cake powder 20-30g, carbamide 0.2-2g, sucrose 1-5g, k2hpo40.5-2.5g、mgso40.1-0.6g、
mnso40.05-0.5g, bubble enemy 0.1-0.5g, ph 7.2, then it is diluted to 1000ml.
Present invention also offers a kind of biocontrol water composite microbiological fertilizer, including above-mentioned biocontrol water complex microorganism bacterium solution and
Liquid organic starting material, described biocontrol water complex microorganism bacterium solution accounts for the 0.1%-3% of gross weight.
The production method of above-mentioned composite microbiological fertilizer is, described bacillus subtilises lvle14 and bacillus amyloliquefaciens
After lvle15 is fermented into bacterium solution respectively, it is mixed to get complex microorganism bacterium solution, by complex microorganism bacterium solution according to 0.1-3% mass
Ratio is added in liquid organic starting material, makes liquid composite microorganism fertilizer.Complex microorganism bacterium solution 0.1-3% is that to account for fertilizer total
The mass ratio of weight.
Preferably, the content of organic matter of described liquid organic starting material is more than 30%；
By above-mentioned bacillus subtilises (bacillus subtilis) lvle14 bacterium solution and bacillus amyloliquefaciens
(bacillus amylolique faciens) lvle15 bacterium solution mixes after adsorbing respectively, obtains biocontrol water composite microbial bacteria
Agent, described composite microbiological fertilizer includes biological control composite microbial agent and the SOLID ORGANIC raw material of 2-10%.2-10%'s
Biological control composite microbial agent is the mass ratio accounting for fertilizer gross weight.
Preferably, the content of organic matter of described SOLID ORGANIC raw material is more than 30%.
Further, described liquid organic starting material is selected from yeast waste liquor or amino acid waste liquid；Described SOLID ORGANIC raw material
Selected from the hydrolysis residue of amino acids production process or the dregs of fat of edible fat production process.
Present invention also offers a kind of production method of biocontrol water composite microbiological fertilizer,
Solid composite microbe fertilizer production: the ph of SOLID ORGANIC raw material is controlled in 5.0-8.0, pulverizes；Will be above-mentioned withered
Careless bacillus cereuss lvle14 and bacillus amyloliquefaciens lvle15 fermenting and producing respectively, gained bacterium solution is adsorbed with adsorbing medium respectively
After mix, obtain biological control composite microbial agent, complex micro organism fungicide be added to solid according to 2%-10% mass ratio
In powdery Organic Ingredientss, obtain solid composite microbe fertilizer.
It is of course also possible to after bacterium solution is first mixed, then with adsorbing medium absorption, obtain biological control composite microbial agent,
Complex micro organism fungicide is added in solid, powdery Organic Ingredientss according to 2%-10% mass ratio, obtains solid composite microbe
Fertilizer.
Preferably, bacillus subtilises lvle14 and bacillus amyloliquefaciens lvle15 in described complex microorganism bacterium solution
Mass ratio be 1-4:1.Bacillus subtilises lvle14 and bacillus amyloliquefaciens lvle15 in described complex micro organism fungicide
Mass ratio be 1-4:1.The quality of bacterium solution and adsorbing medium is than general control in 1:2-6.
It is further preferred that bacillus subtilises lvle14 and bacillus amyloliquefaciens in described complex microorganism bacterium solution
The mass ratio of lvle15 is 2-3:1.It is further preferred that bacillus subtilises lvle14 conciliates shallow lake in described complex micro organism fungicide
The mass ratio of afnyloliquefaciens lvle15 is 2-3:1.
Preferably, can also add in described composite microbiological fertilizer N,P,K fertilizers one or more.
Trace-element fertilizer can also be added as needed.
Present invention also offers above-mentioned biocontrol water complex microorganism bacterium solution especially suppresses anthrax in suppression crop pest
On application.
And provide above-mentioned biocontrol water composite microbiological fertilizer especially suppress on anthrax in suppression crop pest and
Increase the application of fertilizer efficiency.
The main component of preferably several raw materials:
Yeast waste liquor: purchased from Angel Yeast Co., Ltd.Main component is as follows:
Table 1: yeast concentrated solution main nutrient composition table
Hydrolysis residue derives from the production process of the aminoacid such as Gourmet Powder Factory, lysine factory.The base of hydrolysis residue after treatment
This character is as follows:
Table 2: the Elemental characters of hydrolysis residue (powdery) after process
The dregs of fat come from Shanghai roc Environmental Protection Technology Co., Ltd, and main component is as follows:
Table 3: dregs of fat main nutrient composition table
Preferably, can also add in described composite microbiological fertilizer npk fertilizer one or more.As
The inorganic element content of itself, n+p in fruit Organic Ingredientss2o5+k2O ＜ 8%, that is accomplished by suitable interpolation inorganic elementss, preferably
Be n select carbamide supplement, p2o5From be potassium phosphate, k2What o selected is potassium nitrate.Can also be according to soil and crop need
Will, macroelement fertilizer can be added.Trace-element fertilizer can also be added as needed.
Beneficial effects of the present invention
The present invention is a kind of composite microbiological fertilizer with Biological control function and its production method, using rich in organic
The raw material of the crop fertilizer of matter, then make complex microorganism after mixing with the fermentation liquid of Antagonistic Fungi, this product and current city
Product on field is compared and is had the advantage that
1) it is rich in Antagonistic Fungi in fertilizer product, and be compound biocontrol bacterium, its biocontrol effect highly significant is fertile
In material, the content of this bacterial strain is up to 100,000,000/more than g/ml, can form dominant microflora after being manured into soil, and under survival in soil
Come.
2) the preferred content of organic matter of Organic Ingredientss that the present invention selects is higher, and nutrient content is higher, is substantially not required to
Inorganic nutrients to be added.
3), after product of the present invention is combined to two kinds of bacterium, the preventive effect for anthrax has reached 88.3%, and ratio is used alone two
The preventive effect of bacterium is remarkably reinforced.
4) biocontrol water composite microbiological fertilizer has diseases prevention and fertilizer efficiency effect concurrently after applying, and can improve crop resistance, and
Absorption to fertilizer and utilization rate, keep plant growth healthy and strong, and the breeding of biocontrol microorganisms also can be promoted in turn to improve crop disease-resistant
Ability, and contribute to forming good agricultural eco-system.
5) pesticide dosage can be reduced while reducing fertilizer amount, reduce pesticide residues and pollution, for sustainable agriculture
The raising of the development of industry and pollution-free food production and people's living standard has important function.
Specific embodiment
Bacillus amyloliquefaciens strain separation process
1. soil sample collection and process
There is serious field from the Viola grypoceras A. Gray growing area epidemic disease such as Shanghai, Jiashan, Pinghu, using five point sampling methods from growth
Healthy Fructus Cucumidis sativi rhizosphere takes soil sample.Scalp top layer 5cm soil layer during sampling, take fresh soil sample 500g, preserved with freshness protection package.Weigh soil
In the triangular flask equipped with 90ml sterilized water for the sample 10g addition (built-in bead is some), 30 DEG C, 120r/min vibration 30min, standing
Obtain soil supension afterwards.
2. the screening of biocontrol bacteria
Soil supension is diluted to 10-4、10-5、10-6Concentration, draws different dilution soil supension 100 μ l and coats
In na culture medium, it is placed in 30 DEG C of cultures 48 hours, the different antibacterial single bacterium colony such as picking color, form purification, preservation.Using
Flat board face-off method measures the bacteriostatic activity to powdery mildew of cucumber for the antibacterial being separated to.Powdery mildew of cucumber is activated on pda flat board,
Beaten in colony edge with card punch and take diameter 6mm truffle, be inoculated into pda flat board central authorities.Apart from powdery mildew of cucumber after cultivating 1 day
4 docking point inoculation tested bacteria at truffle 2cm, 28 DEG C of cultures, observe antibacterial result after 3-5 days, each process sets 3 weights
Multiple.Choose bacterial strain numbering lvle-15 of good antimicrobial effect.
3. the identification of antagonistic strain
3.1 bacterial strain bio-chemical characteristics: the bacterial strain of the fungistatic effect screening is carried out colony morphological observation, microscope shape
State observation, Gram’s staining, spore staining, hydrolysis starch test, methyl red test (m.r), v.p test, citrate test,
The Main Morphologies such as catalase reaction test, gelatin liquefaction test, mobility test and physiological and biochemical index identification.Preliminary Identification
Lvle-15 belongs to for bacillus amyloliquefaciens.
3.2 bacterial strains 16s rdna sequence analysis identification
By antibacterial is carried out with 16s rdna sequencing and comparative analysiss, result shows, 16s rdna and Xie Dian of lvle-15
The 16s rdna of afnyloliquefaciens has the homology of height, and homology reaches 99%.Comparison result is obtained with cell divide method
Qualification result is consistent.
Bacillus subtilis strain separation process
1. soil sample collection and process
There is serious field from the Viola grypoceras A. Gray growing area epidemic disease such as Shanghai, Jiashan, Pinghu, using five point sampling methods from growth
Healthy Fructus Cucumidis sativi rhizosphere takes soil sample.Scalp top layer 5cm soil layer during sampling, take fresh soil sample 500g, preserved with freshness protection package.Weigh soil
In the triangular flask equipped with 90ml sterilized water for the sample 10g addition (built-in bead is some), 30 DEG C, 120r/min vibration 30min, standing
Obtain soil supension afterwards.
2. the screening of biocontrol bacteria
Soil supension is diluted to 10-4、10-5、10-6Concentration, draws different dilution soil supension 100 μ l and coats
In na culture medium, it is placed in 30 DEG C of cultures 48 hours, the different antibacterial single bacterium colony such as picking color, form purification, preservation.Using
Flat board face-off method measures the bacteriostatic activity to powdery mildew of cucumber for the antibacterial being separated to.Powdery mildew of cucumber is activated on pda flat board,
Beaten in colony edge with card punch and take diameter 6mm truffle, be inoculated into pda flat board central authorities.Apart from powdery mildew of cucumber after cultivating 1 day
4 docking point inoculation tested bacteria at truffle 2cm, 28 DEG C of cultures, observe antibacterial result after 3-5 days, each process sets 3 weights
Multiple.Choose bacterial strain numbering lvle-14 of good antimicrobial effect.
3. the identification of antagonistic strain
3.1 bacterial strain bio-chemical characteristics: the bacterial strain of the fungistatic effect screening is carried out colony morphological observation, microscope shape
State observation, Gram’s staining, spore staining, hydrolysis starch test, methyl red test (m.r), v.p test, citrate test,
The Main Morphologies such as catalase reaction test, gelatin liquefaction test, mobility test and physiological and biochemical index identification.Preliminary Identification
Lvle-14 is bacillus subtilis Pseudomonas.
3.2 bacterial strains 16s rdna sequence analysis identification
By antibacterial is carried out with 16s rdna sequencing and comparative analysiss, result shows, the 16s rdna of lvle-14 and hay
The 16s rdna sequence homology of bacillus cereuss reaches 99%.The qualification result that comparison result is obtained with cell divide method is consistent.
Embodiment 1
1st, microbial inoculum: bacillus subtilises lvle14, strain fermentation culture medium prescription is soluble starch 30g, Huang
Semen Glycines powder 15g, sucrose 5g, peptone 5g, bean cake 3g, k2hpo43g、mgso41g, bubble enemy 0.5g, moisturizing is to 1000 milliliters.Micro- life
In thing bacterium solution, living bacteria count is 9.8 × 109Individual/milliliter.
Bacillus amyloliquefaciens lvle15: strain fermentation culture medium prescription be Semen Maydis powder 25g, soybean cake powder 25g, carbamide 1g,
Sucrose 3g, k2hpo41.2g、mgso40.3g、mnso40.2nd, bubble enemy 0.3g, ph 7.2.In microbial inoculum, living bacteria count is
1.49×1010Individual/milliliter.
Bacterium solution after fermentation is mixed with bacillus amyloliquefaciens lvle15 mass ratio 2:1 by bacillus subtilises lvle14.
2nd, composition of raw materials is joined: adds according to formula as below.Trace element is Borax and chelated zinc, and ratio is 1.5:3.5；Protect
Shield agent is sodium benzoate and molasses, and ratio is 1.5:2.
Table 3 liquid composite microorganism fertilizer raw material adds formula
3rd, inorganic nutrients are mixed with 40% (gross mass) water；Protective agent is added in concentrated solution, again will after stirring
Bacterium solution adds；Finally by the inorganic nutrients of abundant dissolving with containing bacterium concentrate mixing, and add remaining moisture, make liquid and be combined
Microbial manure.Finished product fertilizer all technical is as follows.
Table 4 liquid composite microorganism fertilizer the key technical indexes
4th, liquid composite microorganism fertilizer is through liquid filling device subpackage, sealing, warehouse-in.
Embodiment 2
1st, the preparation of solid microbial: the bacterium solution turf after biocontrol water bacillus amyloliquefaciens lvle15 is fermented
In bacterium solution: the ratio of turf=1:4.5 is adsorbed；Bacterium solution after bacillus subtilises lvle14 is fermented presses bacterium with turf
Liquid: turf 1:4 absorption, two kinds of microbial inoculum 1:1 mixing, after mixing, the living bacteria count amount of composite bacteria agent capable reaches 2 × 109Individual/gram more than,
Water tariff collection is within 30%.
2nd, sample.The hydrolysis residue producing during amino acids production, takes from Henan "Lotus" gourmet powder company limited by shares；
3rd, ph conditioning: bulk hydrolysis residue is broken into into pieces powder or little particle, presses hydrolysis residue: Calx=4:1 with powdered lime
Mixing, stacks 8-12 hour after being pulverized with chain crusher, allows it fully to react；
4th, turn upside down: with forklift or artificial turning, allow reacted temperature of charge cooling reach room temperature；
5th, pulverize: the hydrolysis residue being cooled to room temperature is measured, fundamental property is shown in Table 4, content of beary metal well below
The industry standard of composite microbiological fertilizer, is not measured；
Table 4: the Elemental characters of hydrolysis residue after process
6th, dispensing.Blender adds and is slowly added to powdery hydrolysis residue, and by weight 8% ratio adds preparation
Good complex micro organism fungicide.
7th, mix.Blender stirs 3-5 minute, so that material is sufficiently mixed uniformly.
8th, pulverize.60-80 mesh is reached by the fineness that pulverizer makes material.
9th, packaging, metering, warehouse-in.
Embodiment 3
1st, the preparation of solid microbial: the turf of the bacterium solution after bacillus subtilises lvle14 is fermented is by bacterium solution:
Turf 1:4 adsorbs, and the bacterium solution after biocontrol water bacillus amyloliquefaciens lvle15 is fermented presses bacterium solution with turf: turf=1:5's
Ratio is adsorbed, two kinds of microbial inoculum 2:1 mixing, and after mixing, the living bacteria count amount of composite bacteria agent capable reaches 3 × 109Individual/gram more than, water
Divide within being maintained at 30%.
2nd, sample.Adsorb the dregs of fat of generation in salad oil production process, take from Shanghai roc Environmental Protection Technology Co., Ltd；
3rd, ph conditioning: the bulk dregs of fat are broken into into pieces powder or little particle, presses hydrolysis residue with powdered lime: Calx=4:1 mixes
Close, stack 8-12 hour after being pulverized with chain crusher, allow it fully to react；
4th, turn upside down: with forklift or artificial turning, allow reacted temperature of charge cooling reach room temperature；
5th, pulverize: the hydrolysis residue being cooled to room temperature is measured, fundamental property is shown in Table 4, content of beary metal well below
The industry standard of composite microbiological fertilizer, is not measured；
Table 4: the Elemental characters of the dregs of fat after process
6th, dispensing.Blender adds and is slowly added to the powdery dregs of fat, and by weight 7% ratio addition prepares
Complex micro organism fungicide.
7th, mix.Blender stirs 3-5 minute, so that material is sufficiently mixed uniformly.
8th, pulverize.60-80 mesh is reached by the fineness that pulverizer makes material.
9th, packaging, metering, warehouse-in.
Embodiment 4
1st, the preparation of solid microbial: the turf of the bacterium solution after bacillus subtilises lvle14 is fermented is by bacterium solution:
Turf 1:4 adsorbs, and the bacterium solution after biocontrol water bacillus amyloliquefaciens lvle15 is fermented presses bacterium solution with turf: turf=1:3's
Ratio is adsorbed, absorption latter two microbial inoculum 3:1 mixing, and after mixing, the living bacteria count amount of composite bacteria agent capable reaches 3.8 × 109Individual/
More than gram, water tariff collection is within 30%.
2nd, the hydrolysis Slag treatment during amino acids production is pulverized (with embodiment 2)；
3rd, add in blender and be slowly added to powdery hydrolysis residue, and add and to prepare by weight 6% ratio
Complex micro organism fungicide.
Other are with embodiment 2.
Table 5: the composite microbiological fertilizer product quality testing result that different material produces
Test example
1 experimental strain
1.1 bacillus cereuss
Bacillus amyloliquefaciens lvle15 and bacillus subtilises lvle14
1.2 pathogenic fungi
Anthrax
2. experimental technique
2.1 bacterial strain activation
Screening antibacterial is standby after the activation of na flat board, and pathogenic fungi pda flat board is standby after overactivation.
The preparation of 2.2 spore filtrate
Inoculation spore is in nb culture medium 28 DEG C, 200rpm culture 48h, and room temperature, 5000g centrifugation 5min obtain supernatant.Warp again
Cross 0.22um filter filtration sterilization standby.The ratio of bacillus subtilises lvle14 and bacillus amyloliquefaciens lvle15 is 3:
1.
2.3 detection flat board preparations
Filtrate is added with 1:10 ratio and mixes pour plate, every ware 20ml in 50 DEG C of 10/9 times of concentration pda culture medium.
Nb culture medium is added using 1:10 ratio and substitutes fermentation filtrate as comparison.
2.4 pathogenic fungi inoculations
Beaten with card punch (5mm) and take pathogen dish to be placed in corresponding flat board central authorities, pathogen is connect on flat board for comparison compareing,
Test sets 3 repetitions.
2.5 cultures are observed
Culture dish is placed in culture at 28 DEG C, observed detection in the 3rd day, the 5th day, crossing method measurement cause of disease bacterium colony is straight
Footpath.Calculate meansigma methodss, calculate bacteriostasis rate.
Bacteriostasis rate (%)=[(comparison average diameter-experimental group average diameter)/comparison average diameter] × 100%
3 experimental result tables
Table: different strains compare to the inhibition of anthrax
| Antagonistic Fungi | Bacteriostasis rate (%) |
| Bacillus subtilises lvle14 | 8.7 |
| Bacillus amyloliquefaciens lvle15 | 34.9 |
| Composite bacteria agent capable (bacillus subtilises+bacillus amyloliquefaciens) | 88.3 |
Can be seen that bacillus subtilises lvle14 and the bacillus amyloliquefaciens used by the present invention from above test effect
To anthrax, single suppression ratio is respectively 8.7% and 34.9% to lvle15, but the preventive effect after being combined is 88.3%.
The present invention finds in the antagonism carrying out composite testing and bacteriostatic test, conciliates starch bud containing bacillus subtilises
The composite bacteria agent capable of spore bacillus has highly significant inhibitory action to anthrax.
Upper table can be seen that two plants of bacterium and do not have good fungistatic effect to anthrax respectively, and passing ratio compounds
There is highly significant inhibition together afterwards to anthrax.Its reason may is that, the metabolite of bacillus subtilises is solution
The breeding of bacillus amyloliquefacienses provides some necessary somatomedins, thus promoting bacillus amyloliquefaciens Fast-propagation simultaneously
Produce substantial amounts of antagonistic substance, then can not normally be grown by the pathogenic bacteria that this kind of material leads to anthrax.
The biocontrol water composite microbiological fertilizer of the present invention, after two kinds of microbial inoculum mixing, has prominent inhibition to anthrax,
There is larger dissemination.
Test example:
Citrullus vulgariss field test effect
Test process:
A: mu applies composite microbiological fertilizer substrate 120kg of the biocontrol water that turf produces；
B: mu applies the biocontrol water composite microbiological fertilizer 120kg that turf produces；
C: mu applies biocontrol water composite microbiological fertilizer substrate 120kg that acid hydrolysis slag produces；
D: mu applies the biocontrol water composite microbiological fertilizer 120kg that acid hydrolysis slag produces.
Using the composite microbiological fertilizer in embodiment 4.Fertilizer living bacteria count >=2 × 107/ gram, available nutrient >=
6%；Substrate is that composite microbiological fertilizer sterilizes.Plot area 20m2If, 3 repetitions, random district's groups arrangement.At all tests
Reason all makees base manure, and later stage fertilising and management and habits fertilization are identical.Check significance with duncan's new multiple range method.
The impact to Citrullus vulgariss economical character for table 7 different disposal
As can be seen from Table 7: with acid hydrolysis slag produce composite microbiological fertilizer produce than with turf compound micro-
The Citrullus vulgariss main stem length increase 15-23cm of bio-feritlizer, physiology section position increase 4.8-5.5cm, knot melon number increase 0.03-0.17/
Strain, single melon increases 0.43-0.51kg again, illustrates that the composite microbiological fertilizer that acid hydrolysis slag produces can carry than more comprehensively
For the nutrient needed for watermelon growing hence it is evident that improving single melon weight, make the robust plant of Citrullus vulgariss, blade increases, make to nourish and grow and
Reproductive growth is more rational.Applying fertilizer and administration discrete phase long, physiology section position more climing than the Citrullus vulgariss of fertilizer treatment and single melon weight are all
Higher than substrate, the beneficial microbe in this explanation bio-fertilizer has very strong activity in soil, has obvious fertilizer efficiency effect.
The inhibition to watermelon anthrax for table 8 different disposal
This test is the controlling experiment carrying out than more serious several melon dish protecting field in the generation of Beijing suburb anthrax, applies
All exceeded with the emergence rate of the bio-feritlizer of acid hydrolysis slag production, sick seedling rate, prevention effect and amount of increase in production and utilize turf
The bio-feritlizer producing.Illustrate that the composite microbiological fertilizer that acid hydrolysis slag produces can significantly reduce watermelon anthrax and prevent
The sickness rate of disease, dramatically increases the emergence rate of Citrullus vulgariss, reduces sick seedling rate, thus reaching the purpose of volume increase；Apply what turf produced
Fertilizer and discrete phase ratio, the emergence rate of fertilizer, prevention effect and amount of increase in production are higher than to apply substrate, but the amplitude being to increase is failed to understand
Aobvious, effect is not notable, illustrates that effect in turf for the biocontrol microorganisms is not apparent；Apply acid hydrolysis slag substrate and fertilizer phase
The emergence rate of fertilizer, sick seedling rate, prevention effect and amount of increase in production are above applying substrate to ratio, illustrate that biocontrol microorganisms have in soil
Very strong activity, has significant inhibition to watermelon anthrax diseases prevention.
From above test effect it can be seen that the biocontrol water composite microbiological fertilizer being produced with acid hydrolysis slag can be notable
Improve the fertilizer efficiency of bio-feritlizer, have obvious prevention effect to the disease of crop, greatly reduce sick seedling rate, increasing both production and income same
When, the robust plant of Citrullus vulgariss can be made, leaf than the more comprehensive nutrient providing needed for watermelon growing hence it is evident that improving single melon weight
Piece increases, and makes to nourish and grow and reproductive growth is more rational, on the other hand may also function as twice laid, reduces the wasting of resources, guarantor
Retaining ring border, saving mineral resources, minimizing fertilizer amount, the effect reducing peasant burden, improving quality of agricultural product.
Claims (10)
1. a kind of biocontrol water complex microorganism bacterium solution it is characterised in that: described biocontrol water complex microorganism bacterium solution includes hay bud
Spore bacillus (bacillus subtilis) lvle14 bacterium solution and bacillus amyloliquefaciens (bacillus amylolique
Faciens) lvle15 bacterium solution.
2. biocontrol water complex microorganism bacterium solution according to claim 1 it is characterised in that: in described complex microorganism bacterium solution
The mass ratio of bacillus subtilises lvle14 and bacillus amyloliquefaciens lvle15 is 1-4:1.
3. biocontrol water complex microorganism bacterium solution according to claim 1 it is characterised in that: described bacillus subtilises
The fermentation condition of lvle14 bacterium solution fermenting and producing is: ph6.5-7.5, and cultivation temperature is 20-40 DEG C, initial rotating speed 100-
200rpm, ventilation is 0.5-1vvm, cultivates 35-40 hour, and fermentation middle and late stage row becomes spore, spore rate up to more than 95%,
Living bacteria count in fermentation liquid is in 10,000,000,000/more than ml；
The fermentation condition of described bacillus amyloliquefaciens lvle15 bacterium solution fermenting and producing is: ph is 6.5-8.0, and cultivation temperature is
30-45 DEG C, initial rotating speed 120-200rpm, ventilation is 0.5-1vvm, cultivates 35-45 hour, and fermentation middle and late stage row becomes spore,
Spore rate up to more than 93%, the living bacteria count in fermentation liquid is in 15,000,000,000/more than ml.
4. a kind of biocontrol water composite microbiological fertilizer it is characterised in that: include claim 1 described in biocontrol water composite microbial bacteria
Liquid and liquid organic starting material, described biocontrol water complex microorganism bacterium solution accounts for the 0.1%-3% of gross weight.
5. a kind of biocontrol water composite microbiological fertilizer described in claim 4 production method it is characterised in that: claim 1
After described bacillus subtilises lvle14 and bacillus amyloliquefaciens lvle15 is fermented into bacterium solution respectively, it is mixed to get composite microbial
Thing bacterium solution, complex microorganism bacterium solution is added in liquid organic starting material according to 0.1-3% mass ratio, makes liquid composite microbial
Thing fertilizer.
6. a kind of solid biocontrol water composite microbiological fertilizer it is characterised in that: by bacillus subtilises described in claim 1
(bacillus subtilis) lvle14 bacterium solution and bacillus amyloliquefaciens (bacillus amylolique faciens)
Lvle15 bacterium solution mixes after adsorbing respectively, obtains biological control composite microbial agent, and described composite microbiological fertilizer includes 2-
10% biological control composite microbial agent and SOLID ORGANIC raw material.
7. biocontrol water composite microbiological fertilizer described in claim 6 production method it is characterised in that:
The ph of SOLID ORGANIC raw material is controlled in 5.0-8.0, pulverizes；By bacillus subtilises lvle14 described in claim 1 and
Bacillus amyloliquefaciens lvle15 fermenting and producing respectively, gained bacterium solution is mixed with after adsorbing medium absorption respectively, obtains biocontrol water
Complex micro organism fungicide, complex micro organism fungicide is added in solid, powdery Organic Ingredientss according to 2%-10% mass ratio, obtains
To solid composite microbe fertilizer.
8. biocontrol water composite microbiological fertilizer according to claim 6 production method it is characterised in that: described compound micro-
Can also add in bio-feritlizer N,P,K fertilizers one or more.
9. biocontrol water complex microorganism bacterium solution described in claim 1 especially suppresses answering on anthrax in suppression crop pest
With.
10. biocontrol water composite microbiological fertilizer described in claim 4 or 6 suppression crop pest especially suppress on anthrax with
And increase the application of fertilizer efficiency.
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| CN201610756759.1A CN106367367A (en) | 2016-08-29 | 2016-08-29 | Biocontrol compound microbial liquid, compound microbial fertilizer and production method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106977301A (en) * | 2017-04-28 | 2017-07-25 | 上海诺同农业科技有限公司 | A kind of protected fruit trees infiltrating irrigation special bio liquid fertilizer, production method and application |
| CN113966747A (en) * | 2021-06-30 | 2022-01-25 | 中国热带农业科学院环境与植物保护研究所 | A kind of nano-silver composite Bacillus inoculant for preventing and controlling the fruit rot and anthracnose of the fruit of lotus mist fruit and preparation method thereof |
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| CN103865843A (en) * | 2014-02-13 | 2014-06-18 | 河北省科学院生物研究所 | Compound microbial bacteria and application thereof in prevention and treatment of vegetable fungal diseases |
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| CN106977301A (en) * | 2017-04-28 | 2017-07-25 | 上海诺同农业科技有限公司 | A kind of protected fruit trees infiltrating irrigation special bio liquid fertilizer, production method and application |
| CN113966747A (en) * | 2021-06-30 | 2022-01-25 | 中国热带农业科学院环境与植物保护研究所 | A kind of nano-silver composite Bacillus inoculant for preventing and controlling the fruit rot and anthracnose of the fruit of lotus mist fruit and preparation method thereof |
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Application publication date: 20170201 |