CN105950519A - Complex fungicide and preparing and application thereof - Google Patents

Complex fungicide and preparing and application thereof Download PDF

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Publication number
CN105950519A
CN105950519A CN201610559353.4A CN201610559353A CN105950519A CN 105950519 A CN105950519 A CN 105950519A CN 201610559353 A CN201610559353 A CN 201610559353A CN 105950519 A CN105950519 A CN 105950519A
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liquid
agent capable
bacillus amyloliquefaciens
composite bacteria
bacteria agent
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张传军
张晓红
张中亲
白玉红
李冰洁
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Beijing Kanion Yisheng Biological Technology Co Ltd
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Beijing Kanion Yisheng Biological Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • 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
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Abstract

The invention relates to a microorganism fungicide, in particular to a complex fungicide and preparing and application thereof. Bacillus amyloliquefaciens includes CGMCCNO.12306(KY-1), CGMCCNO.12307(KY-2) and CGMCCNO.12308(KY-3) respectively, and the CGMCCNO.12306(KY-1), the CGMCCNO.12307(KY-2) and the CGMCCNO.12308(KY-3) are collected in China General Microbiological Culture Collection Center. The bacillus amyloliquefaciens is subjected to three-grade liquid fermentation respectively, and then the fermentation liquid is mixed according to the volume ratio of the CGMCCNO.12306(KY-1) to the CGMCCNO.12307(KY-2) to the CGMCCNO.12308(KY-3) of (5-1):(1-5):(5-1) to obtain the complex fungicide. According to the microorganism fungicide, the bacillus amyloliquefaciens KY-1, the bacillus amyloliquefaciens KY-2 and the bacillus amyloliquefaciens KY-3 which are obtained according to rhizosphere soil of different crops in different territories have different physiological functions, and antagonism is not generated between the bacillus amyloliquefaciens KY-1, the bacillus amyloliquefaciens KY-2 and the bacillus amyloliquefaciens KY-3.

Description

A kind of composite bacteria agent capable and preparation and application thereof
Technical field
The present invention relates to microbial bacterial agent, specifically a kind of composite bacteria agent capable and preparation and application thereof.
Background technology
Soil-borne disease refer to the pathogen living in soil when condition is suitable from crop root or stem foot The disease that portion encroaches on crop and causes.Common are damping off, damping-off, droop, verticillium wilt, Soft rot, root rot, epidemic disease, black rot, sclerotiniose, root knot nematode disease etc..This type of disease in by The trend that year increases the weight of.The direct economic loss caused to agricultural production every year reaches 40%-60%, and serious reaches More than 80%.The development of serious engagement device agricultural.
The prevention and controls of this disease mainly includes crop rotation, cultivates disease-resistant variety and use chemical agent at present. Wherein crop rotation diseases prevention is widely used, but some special economic crops or particular locality are unable to reach conjunction The requirement of reason crop rotation, and have bigger limitation；Conventional breeding for disease resistance is asked of both existing Inscribing and make slow progress, one is to utilize single resistant gene that soil-borne disease pathogen is developed into newly Microspecies, two is that anti-source material scarcity makes conventional breeding can not meet the needs in production, and utilizes existing The breeding work carrying out anti-soil-borne disease for biotechnology is the most still in the exploratory stage.Chemical agent due to The Expected Results got instant result can be received, be also widely used, but the life-time service band of chemical agent Carry out a series of side effect, destroyed ecological balance, cause Minor diseases rampant, the residual of medicament Cause the pollution of water source soil.
Simultaneously because pursue high crop yield simply, it is excessively used chemical fertilizer so that soil compaction, soil ground Power declines, and quality of agricultural product declines, and in fruit and vegerable, the nitrate content problem such as exceed standard is increasingly severe.With Expanding economy and growth in the living standard, the high-quality of agricultural product and safety in production are increasingly by society The extensive concern of meeting.
Microbial manure, by the effect of its uniqueness, is gradually applied to agricultural production.Microbial manure Middle beneficial microbe can produce glucide, accounts for the 0.1% of the soil organism, with plant mucilage, mineral Idiosome and organic colloid combine, and can improve soil aggregate, strengthen the physical property of soil The loss of soil particle and can be reduced, under certain conditions, moreover it is possible to participate in humus and formed.Improve Soil physical property, is conducive to increasing soil fertility.
It is with fertilizer or the reasonably combined use of chemical fertilizer, it is achieved balance fertilizing, thus improves chemical fertilizer Utilization rate, reduces the amount of application of chemical fertilizer, cost-effective, reduces and pollutes；
The microbial manure being made up of the strain with specific function, not only reduces pollution of agricultural products, changes The quality of kind agricultural product, the aspect of preventing and treating in soil-borne disease plays very important effect.
Antibacterial conventional in the preventing and treating of soil-borne disease is that Bacillius belongs to (spore genus).It is that a class is wide The general saprophytic bacteria being present in nature, growth is fast, nutrition is simple, it is possible to formation has the most degeneration-resistant The spore of ability.Be currently used for plant disease mainly have B.subtilis (bacillus subtilis), B.cereus (Bacillus cereus).
Showing according to data of literatures, bacillus amyloliquefaciens is a kind of and bacillus subtilis affinity The highest antibacterial, produces a series of metabolite in the growth course of himself, these secondary generations Thanking to product makes bacillus amyloliquefaciens can extensively suppress the activity of fungus and antibacterial, and promotes plant Growth, prevents and treats root-knot nematode.
Result of study shows, Biocontrol Bacillus press down disease, growth promotion mechanism mainly has four kinds: one, Produce antibiotic agents, directly suppress growth of pathogenic bacteria；Two, secretion growth hormones material, promotees Enter plant growing；Three, activation nutrient, needed for plant growing；Four, induction plant produces disease resistance. But use not pertinent literature report for the mixing of many bacterial strains is compounding.
Summary of the invention
It is an object of the present invention to provide a kind of composite bacteria agent capable and preparation and application thereof.
For achieving the above object, the present invention uses the technical scheme to be:
A kind of bacillus amyloliquefaciens, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) It is respectively as follows: CGMCCNO.12306 (KY-1), CGMCCNO.12307 (KY-2), CGMCCNO.12308 (KY-3), China Committee for Culture Collection of Microorganisms's common micro-organisms center all it is preserved in, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date is on March 24th, 2016.
Wherein, spring stubble planted by bacterial strain is planted from Beipiao, liaoning land 13 years green apple trees, East Liaoning's canopy Three kinds of bacillus cereuss of separation screening in the rhizosphere soil of Fructus Lycopersici esculenti and Jilin Song Yuan Panax Ginseng are logical Cross and its 16S gene order and the date comprision in GenBank are found it and solves starch spore bar Bacterium (Bacillus amyloliquefaciens) similarity is the highest, is 99.9%, its morphological characteristic and Physio-biochemical characteristics are most like with bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Thus the KY-1KY-2KY-3 separated is confirmed as bacillus amyloliquefaciens.
The bacterium colony kenel of above-mentioned bacterial strains is on nutrient agar panel, 37 DEG C, cultivates 96 hours, belongs to and holding concurrently Property anaerobism.Bacterium colony is faint yellow opaque, rough surface, and edge is irregular, and zigzag has protuberance, The most chromogenic element.
The strain of above-mentioned preservation is inoculated in slant medium, in 37 DEG C of constant temperature quiescent culture 24h, microscopy It is more than 95% without miscellaneous bacteria and spore rate, and in 4 DEG C of Storage in refrigerator.Use in 1 year.
The inclined-plane formula (weight ratio) used: glucose 0.1%～4.00%, yeast leaching powder 0.01%～ 1.50%, peptone 0.01%～1.50%, Carnis Bovis seu Bubali cream 0.01%～1.00%, sodium chloride 0.01%～1.00%, Calcium chloride 0.01%～1.00%, disodium hydrogen phosphate 0.01%～1.00%, soluble starch 0.02%～ 2.00%, ammonium sulfate 0.01%～1.00%, manganese sulfate 0.1mg/L～10mg/L, sodium molybdate 0.1 Mg/L～10mg/L, agar powder 1.2%～2.5%, surplus are water.PH=7.1-7.2.121 DEG C, go out Bacterium 30min.
The excellent slant strains of preservation, is inoculated in liquid bottles culture medium, in 180rpm, and 30 DEG C～32 DEG C, constant-temperature shaking culture 20h～24h, obtain a bottle liquid spawn.
The liquid bottles formula (weight ratio) used: glucose 0.20%～5.00%, soybean cake powder 0.10%～ 4.00%, dipotassium hydrogen phosphate 0.02%～1.00%, yeast leaching powder 0.02%～3.00%, magnesium sulfate 0.02%～ 2.00%, peptone 0.02%～2.00%, sucrose 0.02%～4.00%, Semen Maydis pulp 0.02%～4.00%, Carbamide 0.02%～4.00%, Nacl 0.02%～1.00%, calcium carbonate 0.02%～2.00%, bubble enemy 0.1%～0.4%, surplus is water.PH=7.0-7.4.121 DEG C of sterilizing 30min.
A kind of composite bacteria agent capable, by bacillus amyloliquefaciens described above respectively through tertiary liquid fermentation, The most by volume (5～1): (1～5): fermentation liquid mixing is i.e. obtained composite bacteria agent capable by (5～1) ratio.
Spore rate in above-mentioned bacterial strains fermentation liquid after tertiary liquid fermentation respectively in described composite bacteria agent capable >= 90%, blood cell count plate >=10,000,000,000/ml, residual glucose≤0.6%, pH=7.0～8.0.
The mixed fermentation liquid that will mix according to the above ratio adds mixed fermentation liquid and amasss 0.1～2.0% Stabilizer, i.e. obtains liquid preparation.
The mixed fermentation liquid mixed according to the above ratio is amassed through falling film concentration to mixed fermentation liquid 1/2-1/3, then adds mixed fermentation liquid after concentrating and amasss 0.1～the stabilizer of 2.0% and mixed after concentrating Close the carrier of fermentating liquid volume 30%～40%, be sufficiently mixed after addition, be spray-dried after mixing, to obtain final product Compound bacteria powder.
Described stabilizer is sodium benzoate and/or aminoacid；Carrier be bentonite, powdered rice hulls, zeolite powder, Several mixing in white carbon, calcium carbonate；Wherein, aminoacid is lysine.
A kind of preparation method of composite bacteria agent capable:
The liquid seeds liquid of 3 kinds of bacillus amyloliquefaciens is seeded to first order seed by 0.5%～1% respectively In tank culture medium, in 30 DEG C～32 DEG C, tank pressure=0.02MPa～0.05MPa, ventilation ratio=1:0.5～1: 0.8, first class seed pot cycle=12h～24h, carry out one grade fermemtation cultivation, to Biomass >=10%, bacterium Volume morphing is consistent, and rod-short, two ends are concordant, colour homogeneous, and pH gos up naturally to 6.5～7.0, treats With；Wherein, utilize flame inoculation method access certain volume without miscellaneous bacteria and standard compliant bottle kind Sub-liquid.
According to secondary seed tank culture medium dispensing, sterilizing in secondary seed tank, simultaneously by grain conductor road Sterilizing (123 DEG C of sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, Sending out without miscellaneous bacteria and standard compliant above-mentioned one-level of 1-15% volume is accessed by aseptic seed transferring pipeline Ferment culture fluid, in 30 DEG C～32 DEG C, tank pressure=0.02MPa～0.05MPa, ventilation ratio=1:0.5～1: 0.8, secondary seed tank cultivation cycle=12h～24h, carry out second order fermentation cultivation, to Biomass >=10%, Thalli morphology is consistent, rod-short, and two ends are concordant, colour homogeneous, and pH gos up naturally to 6.5～7.0, Stand-by；
According to fermentation tank culture medium dispensing, sterilizing in fermentation tank, simultaneously by grain conductor road sterilizing (123 DEG C sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, by aseptic Seed transferring pipeline access 2-15% volume without miscellaneous bacteria and standard compliant above-mentioned second order fermentation culture fluid, In 30 DEG C～32 DEG C, tank pressure=0.04MPa～0.07MPa, ventilation ratio=1:0.8～1:1.2, pass through It is that 20%～25% ammonia controls pH=6.5-6.7 that stream adds concentration, until pH gos up naturally, passes through simultaneously The glucose solution that stream adds 80%, maintains concentration of glucose >=1% of residual, cycle in sweat =30h～72h；When spore rate is more than 90%, and viable count is more than 10,000,000,000/ml, residual sugar≤0.6%, terminates Fermentation, obtains three grade fermemtation liquid the most respectively.
Wherein, in joining sugar bowl, prepare the glucose solution of 80% and in 115 DEG C, 20 minutes sterilizations, It is cooled to 30 DEG C, stand-by；Addition is 10L～15L per minute.
Then according to volume ratio=(5～1): (1～5): the ratio of (5～1), by fermentation liquid mixing i.e. Obtain composite bacteria agent capable.
The mixed fermentation liquid that will mix according to the above ratio adds mixed fermentation liquid and amasss the steady of 0.1-2.0% Determine agent, i.e. obtain liquid preparation.
By the mixed fermentation liquid that mixes according to the above ratio through the 1/2 1/3 of falling film concentration to mixed volume, Then add the stabilizer of volume 0.1-2% after concentrating, add the carrier of volume 30%～40% after concentration It is sufficiently mixed, is spray-dried and obtains compound bacteria powder.
Described stabilizer is sodium benzoate and/or aminoacid；Carrier be bentonite, powdered rice hulls, zeolite powder, Several mixing in white carbon, calcium carbonate；Wherein, aminoacid is lysine；Described stabilizer is Sodium benzoate and aminoacid, both mass ratioes are (2～5): (1～10).
Described carrier is bentonite, powdered rice hulls, zeolite powder, white carbon and calcium carbonate, is in mass ratio Bentonite: powdered rice hulls: zeolite powder: white carbon: calcium carbonate=(20～40): (2～10): (20～ 35): (1～10): the ratio mixing of (10～30).
(1) one-level and secondary seed tank culture medium prescription
Glucose 0.20%～5.00%, soybean cake powder 0.10%～4.00%, dipotassium hydrogen phosphate 0.02%～ 1.00%, yeast leaching powder 0.02%～3.00%, magnesium sulfate 0.02%～2.00%, Peptone 0.02%～2.00%, sucrose 0.02%～4.00%, Semen Maydis pulp 0.02%～4.00%, Carbamide 0.02%～4.00%, Nacl 0.02%～1.00%, calcium carbonate 0.02%～2.00%, bubble enemy 0.1%～0.4%, surplus is water.PH=7.0-7.4.121 DEG C of sterilizing 30min.
(2) fermentor cultivation based formulas
Glucose 0.10%～6.00%, soybean cake powder 0.10%～4.00%, Semen Maydis powder 0.10%～4.00%, Peanut cake powder 0.10%～4.00%, cottonseed meal 0.10%～4.00%, Semen Maydis pulp 0.10%～3.00%, Yeast leaching powder 0.10%～2.00%, peptone 0.10%～3.00%, Nacl 0.10%～2.00%, carbon Acid calcium 0.10%～2.00%, surplus are water.PH=6.5-7.4.121 DEG C of sterilizing 30min.
Beneficial effects of the present invention:
The present invention is according to the three strain solution starch buds obtained in the rhizosphere soil of different geographical different crops Spore bacillus, KY-1, KY-2, KY-3, they have different physiological functions, and do not produce Raw Antagonism.
Wherein, bacterial strain KY-1 cultivates on egg yolk agar plate, it is possible to produce transparent hydrolysis circle, should Bacterial strain has dissolving phosphor and dissolving potassium function, dissolution of minerals phosphorus and other nutrient substance and promotes plant growing.Bacterial strain KY-2 is to utilize eelworm-killing activity high frequency zone model discrimination to obtain, the root knot line to various vegetables melon and fruit Worm, has good preventive and therapeutic effect, and to trialeurodes vaporariorum, bollworm, oriental tobacco budworm, diamondback moth also has very well Avoidance effect.Bacterial strain KY-3 can produce bigger transparent hydrolysis circle on Screening Protease flat board, On the qualification flat board identify bacterium, producing transparent inhibition zone with escherichia coli and aspergillus niger simultaneously, right The disease of the bacillary and fungoid of multiple kinds of crops has good preventive and therapeutic effect.
Above-mentioned three bacillus amyloliquefaciens are used fermentation technology to obtain microbial bacterial agent, bacterium by the present invention Agent is made up of the microbial cells lived and metabolite thereof, as various organized enzymes, antibacterial peptide, lipopeptid with And phytohormone class, biological activity is high；Viable count is high, enters Fast-propagation after soil, increases rhizosphere The microorganism species of soil, improves physical property and the crumb structure of soil, alleviates soil compaction, promotees Entering the decomposition of organic substance, constantly secrete secondary metabolite simultaneously, the physiological vital of regulation plant is lived Dynamic, prevent and treat generation and the propagation of soil-borne disease, increasing both production and income, improve quality of agricultural product.At agricultural product The diseases prevention in storage after harvesting, fresh-keeping also there is certain effect.
Specifically, it is thus achieved that the microbial inoculum of powder body, there is higher viable count, be suitable for and base fertilizer, the most excellent The farm manure of matter is used in mixed way.After being manured into soil, accommodative ability of environment is strong, and reproduction speed is fast, is increasing While adding beneficial rhizosphere microorganisms flora, decompose rapidly organic matter and utilize for plant absorption, improve soil Earth fertility, strengthens soil infiltration, water conservation and breathability, promotes root system fast-growth, prevent soil-borne disease Spreading of evil.
Obtain liquid preparation, be mainly used in foliage-spray, insect protected, cure the disease, promote Chlorophyll synthesis, Strengthen photosynthesis, accelerate plant metabolism and protein synthesis, promote early ripening of the crops to get bumper crops, increase With the sugar content of melon and fruit, improve the anti-ageing cold tolerance of crop disease-resistant, improve nutrient quality.
Utilize the microbial inoculum of above-mentioned acquisition keeping ecological balance, reduce cost while to booth vegetable, Melon and fruit, the various plants such as field crop, Chinese crude drug and landscape flower all has good growth promotion, increasing Product, diseases prevention, insect protected, the effect of raising fruit quality, and safe to the human body, free from environmental pollution.
Detailed description of the invention
Microbial-bacterial fertilizer of the present invention, is respectively through tertiary liquid fermentation by three bacillus amyloliquefaciens Reach to put tank standard, then the fermentation liquid of three bacillus amyloliquefaciens of above-mentioned fermentation gained mixed, Process through special again.Three described bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) CGMCCNO.12306 (KY-1), CGMCCNO.12307 it are respectively as follows: (KY-2), CGMCCNO.12308 (KY-3), be all preserved in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date It it is on March 24th, 2016.
Below in conjunction with being embodied as case, the present invention will be further described
Embodiment 1
The acquisition of bacterial strain:
1. the collection of soil sample
(1) after summer, rainwater.Crop rhizosphere Tu Biao lower floor about 5 centimetres～10 centimetres In the range of gather soil sample；
(2) diseased region is selected, but the rhizosphere soil under the individual plants do not fallen ill；
(3) it is preserved in aseptic ventilative plastic bag.
2. enrichment culture
(1) prepared by culture medium
General nutrition agar plate g/L: Carnis Bovis seu Bubali cream 3.0, peptone 10.0, sodium chloride 5.0, pure Water purification 1000ml, agar powder 2.0, pH=7.0～7.2, nystatin 50mg/L
(2) soil sample processes
Weigh soil sample 1g, join in the sterilized water of 99ml, agitator vibration 15min.In aseptic bar Filter under part, obtain filtrate
(3) with sterilized water by above-mentioned filtrate, according to ten times of gradient dilution methods, 10 it are diluted to one by one-3、10-4、 10-5、10-6
(4) by above-mentioned diluent in 80 DEG C of water bath heat preservation 10min, General nutrition agar plate is coated.Often Individual flat board adds 0.1ml diluent.In 37 DEG C of constant temperature culture 96h
(5) under aseptic condition, each single bacterium colony is coated with biochemical sheet, uses Gram staining method to show Micro mirror is observed.
(6) spore will be produced, and be gram-positive bacterium colony switching ordinary nutrient agar slant, in 37 DEG C of perseverances Temperature cultivates 96h (Medium Proportion is with General nutrition agar plate g/L)
3, line purification: utilize General nutrition agar plate, uses plate streak by oblique for the single bacterium colony obtained Face culture continues purification, until obtaining single typical colonial morphology.Colony morphology characteristic is: Faint yellow opaque, rough surface, edge is irregular, and zigzag has protuberance, the most chromogenic element
4, directed screening
(1) the ability screening of organophosphor and Phos is decomposed
Egg plate g/L: Carnis Bovis seu Bubali cream 3.0, peptone 10.0, sodium chloride 5.0, pure water 1000ml Agar powder 2.0, pH=7.0～7.2, (1 egg yolk adds 20ml sterilized water to aseptic yolk solution 10ml Make sterile solution)
Phos synthetic medium g/L: ammonium sulfate 0.5, magnesium sulfate 0.3, calcium phosphate 10, sodium chloride 0.3, glucose 10g, 1% manganese sulfate 1ml, 1% ferrous sulfate 1ml, pure water 1000ml, agar 20g, pH=7.0～7.5
All bacillus cereuss dibbling respectively after purification is put down in egg plate and Phos synthetic medium Plate, in 37 DEG C of constant temperature culture 96 hours, selects to produce hydrolysis on two kinds of culture plates simultaneously Bacterium colony slant culture carry out preservation.(lyophil preservation: numbered KY-1)
(2) eelworm-killing activity high frequency zone
According to " microorganism journal " 54 (5): 589～594 4 May
2014；It is secondary that " bacillus amyloliquefaciens eelworm-killing activity high frequency zone model " executes intelligent Sun Fan Liu Zhong
Zhang Keqin Huang Wei dawn；Yunnan University's living resources conservation is in utilizing key lab
Document is reported, operates.The bacterial strain selecting eelworm-killing activity high carries out lyophil preservation, numbering For KY-2.
(3) fungus, bacterial resistance screening and proteinase activity screening
Fungus resistant screening culture medium g/L:PDA flat board
Rhizoma Solani tuber osi 200g, glucose 20g, agar powder 15g, pure water 1000ml, pH=6.0. In 121 DEG C, sterilizing 30min.Standby.
Above-mentioned culture medium is cooled to 40-50 DEG C, under aseptic condition, pours the training of a diameter of 90mm into Supporting in ware, every ware adds 20ml.After solidification, then pour containing Aspergillus niger spores that (concentration is thereon into 105) upper strata culture medium 5ml, mix rapidly bacterium, solidify to obtain fungus resistant screening flat board.
Bacterial resistance screening culture medium g/L: plain agar flat board
Carnis Bovis seu Bubali cream 3.0, peptone 10.0, sodium chloride 5.0, pure water 1000ml, agar powder 2.0, PH=7.0～7.2.In 121 DEG C, sterilizing 30min.Standby.
Above-mentioned culture medium is cooled to 40-50 DEG C, under aseptic condition, pours the training of a diameter of 90mm into Supporting in ware, every ware adds 20ml.After solidification, (concentration is 10 pouring into containing escherichia coli on it again5) Upper strata culture medium 5ml, mix rapidly bacterium, solidify to obtain bacterial resistance screening flat board.
Proteinase activity screening flat board g/L:
Carnis Bovis seu Bubali cream 3.0, peptone 10.0, sodium chloride 5.0, pure water 1000ml, agar powder 2.0, Germfree defatted milk 15ml, pH=7.0～7.2.In 121 DEG C, sterilizing 30min.Standby.
Above-mentioned culture medium is cooled to 40-50 DEG C, under aseptic condition, pours the training of a diameter of 90mm into Supporting in ware, every ware adds 20ml.After solidification, obtain Screening Protease flat board.
The sporiferous single bacterium colony slant culture obtained by purification dibbling the most simultaneously is to above-mentioned three kinds of sieves Select on flat board, in 37 DEG C of constant temperature culture 96 hours.It is chosen on these three screening flat board and produces thoroughly simultaneously Single bacterium colony slant culture of open fire Xie Quan carries out lyophil preservation.Numbered KY-3.
5, liquid culture feature
Fluid medium g/L: Carnis Bovis seu Bubali cream 3.0, peptone 10.0, sodium chloride 5.0, pure water 1000ml, PH=7.0～7.2.In 121 DEG C, sterilizing 30min.Standby.
Aseptically, the slant culture 2cm of KY-1, KY-2, KY-3 is taken2, access 100ml In aforesaid liquid culture medium, quiescent culture.Result:
Mycoderma is had to be formed during standing；Gram’s staining is positive.Microscope inspection is shaft-like, endogenous spore, Ovalize, the blunt circle in two ends, sporangium do not expands, and middle life is raw to time end, has motor type.
6, biochemical characteristic
Hydrolysis starch and gelatin, voges-Proskauer test (V-P) is negative, nitrate reduction test is cloudy Property, and phenylalanine deaminase test, indole test, methyl red test, hydrogen sulfide production test be Negative.
Embodiment 2
The preparation of composite bacteria agent capable:
1. the incubation of three bacillus amyloliquefaciens comprises the following steps
1) preparation of solid slope strain
The strain of 3 kinds of preservations is inoculated in slant medium respectively, in 37 DEG C of constant temperature quiescent culture 24h, Microscopy is more than 95% without miscellaneous bacteria and spore rate, and in 4 DEG C of Storage in refrigerator.Use in 1 year.
The inclined-plane formula (weight ratio) used: glucose 0.1%, yeast leaching powder 0.5%, peptone 0.1%, Carnis Bovis seu Bubali cream 0.5%, sodium chloride 0.02%, calcium chloride 0.01%, disodium hydrogen phosphate 0.04%, solubility are formed sediment Powder 1.4%, ammonium sulfate 0.01%, manganese sulfate 0.1mg/L, sodium molybdate 0.1mg/L, agar powder 1.5%, Surplus is water.PH=7.1.121 DEG C, sterilizing 30min.
2) preparation of bottle liquid spawn
By the fresh slant culture of the excellent 3 kind strain of above-mentioned acquisition preservation, it is inoculated in liquid respectively In bottle culture medium, inoculum concentration is 2cm2/100ml.In 180rpm, 30 DEG C～32 DEG C, constant temperature oscillation Cultivate 21h, obtain respective bottle liquid spawn respectively.
The liquid bottles formula (weight ratio) used: glucose 2.00%, soybean cake powder 2.00%, phosphoric acid Hydrogen dipotassium 0.04%, yeast leaching powder 0.5%, magnesium sulfate 0.02%, peptone 0.2%, sucrose 2.00%, Semen Maydis pulp 1.00%, carbamide 0.2%, Nacl 0.02%, calcium carbonate 0.02%, bubble enemy 0.1%, surplus For water.PH=7.4.121 DEG C of sterilizing 30min.
3) fermentative medium formula during tertiary liquid fermentation described in is weight ratio
(1) one-level and secondary seed medium formula
Glucose 2.00%, soybean cake powder 2.00%, dipotassium hydrogen phosphate 0.04%, yeast leaching powder 0.5%, sulphuric acid Magnesium 0.02%, peptone 0.2%, sucrose 2.00%, Semen Maydis pulp 1.00%, carbamide 0.2%, Nacl 0.02%, calcium carbonate 0.02%, bubble enemy 0.1%, surplus is water.PH=7.4.
121 DEG C, sterilizing 30min.
(2) fermentative medium formula
Glucose 3.00%, soybean cake powder 4.00%, Semen Maydis powder 2.00%, peanut cake powder 0.50%, cottonseed meal 0.10%, Semen Maydis pulp 0.55, yeast leaching powder 0.75%, peptone 0.16%, Nacl 0.34, carbonic acid Calcium 0.23%, surplus are water.PH=6.5.121 DEG C of sterilizing 30min.
4) tertiary liquid fermentation comprises the following steps:
3 kinds of bottle liquid spawns of above-mentioned acquisition are carried out tertiary liquid fermentation respectively, specific as follows:
(1) prepared by first class seed pot seed
According to first class seed pot culture medium dispensing, sterilizing in first class seed pot.When tank temperature drop to 32 DEG C During left and right, culture volume is about 200L, utilize flame inoculation method access 1000ml without miscellaneous bacteria and Standard compliant bottle seed liquor.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.02MPa～ 0.05MPa, ventilation ratio=1:0.5～1:0.6；The cultivation cycle of three strain strains is respectively as follows: KY-1 14.5h、KY-2 12.5h、KY-1 16.5h。
(2) prepared by secondary seed tank seed
According to secondary seed tank culture medium dispensing, sterilizing in secondary seed tank, simultaneously by grain conductor road Sterilizing (123 DEG C of sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, Culture volume is about 2000L, by aseptic seed transferring pipeline access 200L without miscellaneous bacteria and meet The first class seed pot seed liquor of standard.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.03MPa～ 0.05MPa, ventilation ratio=1:0.5～1:0.6；The cultivation cycle of three strain strains be respectively as follows: KY-1 12h, KY-2 13h、KY-3 12h。
(3) prepared by fermentation tank
According to fermentation tank culture medium dispensing, sterilizing in fermentation tank, simultaneously by grain conductor road sterilizing (12 3 DEG C of sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, culture medium Volume is about 20000L, by aseptic seed transferring pipeline access 2000L without miscellaneous bacteria and conformance with standard Secondary seed tank seed liquor.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.04MPa～0.07 MPa, ventilation ratio=1:0.8～1:1.2.Control pH=6.5-6.7 by Feeding ammonia water, pass through simultaneously The glucose solution that stream adds 80%, controls residual sugar >=1%, until pH gos up (pH >=7.5) naturally, stops Only Feeding ammonia water and glucose solution, continues to cultivate, until spore rate is more than 90%, terminates fermentation. Wherein, strain KY-1 fermentation: cycle=38h, viable count (plate count)=145.2 hundred million/ml, residual sugar =0.23%, put tank volume=28.3T；Strain KY-2 ferments: cycle=33h, viable count (plate count )=107.4 hundred million/ml, residual sugar=0.13%, put tank volume=25.7T；Strain KY-3 ferments: cycle=40. 5h, viable count (plate count)=115.6 hundred million/ml, residual sugar=0.22%, put tank volume=26.4T.
5) making of NEW TYPE OF COMPOSITE bacterial manure
(1) liquid preparation: meeting of above-mentioned fermentation gained is put three bacillus amyloliquefaciens of tank standard Fermentation liquid, is mixed in isopyknic ratio, cumulative volume about 6T.Mixed liquor cumulative volume 0.5% is added after mixing Stabilizer, detection viable count (plate count)=122.8 hundred million/ml.Fill, warehouse-in.Wherein, surely Determining agent is sodium benzoate and aminoacid, and the mass ratio of the two is 2:1, and aminoacid is lysine；
(2) prepared by powder: meeting of above-mentioned fermentation gained is put the residue three strain solution starch spore bar of tank standard The fermentation liquid of bacterium, is mixed in isopyknic ratio, suitably concentrates through falling film concentration plants, adds and concentrates The stabilizer of rear mixed liquor cumulative volume 0.5%, after adding concentration, mixed liquor cumulative volume 32%% carrier is abundant Mixing, is spray-dried and obtains.Detection viable count (plate count)=235.8 hundred million/ml, packaging, warehouse-in. Described carrier is bentonite, powdered rice hulls, zeolite powder, white carbon and calcium carbonate, in mass ratio 20:30: 20:20:10 ratio mixes.Wherein, stabilizer is sodium benzoate and aminoacid, the mass ratio of the two For 2:1, aminoacid is lysine.
Embodiment 3
Difference from Example 2 is
1, the preparation of solid slope strain
The strain of preservation is inoculated in slant medium, and in 37 DEG C of constant temperature quiescent culture 24h, microscopy is without miscellaneous bacteria And spore rate is more than 95%, and in 4 DEG C of Storage in refrigerator.Use in 1 year.
The inclined-plane formula (weight ratio) used: glucose 0.1%, yeast leaching powder 0.5%, peptone 0.3%, Carnis Bovis seu Bubali cream 0.3%, sodium chloride 0.2%, calcium chloride 0.01%, disodium hydrogen phosphate 0.02%, solubility are formed sediment Powder 2.0%, ammonium sulfate 0.01%, manganese sulfate 0.4mg/L, sodium molybdate 0.2mg/L, agar powder 2.0%, Surplus is water.PH=7.1.121 DEG C, sterilizing 30min.
2, the preparation of bottle liquid spawn
The excellent slant strains of preservation, is inoculated in liquid bottles culture medium, in 180rpm, 30 DEG C～ 32 DEG C, constant-temperature shaking culture 23h, obtain a bottle liquid spawn.
The liquid bottles formula (weight ratio) used: glucose 3.30%, soybean cake powder 1.80%, phosphoric acid Hydrogen dipotassium 0.04%, yeast leaching powder 0.5%, magnesium sulfate 0.04%, peptone 0.5%, sucrose 1.60%, Semen Maydis pulp 1.10%, carbamide 0.12%, Nacl 0.22%, calcium carbonate 0.42%, bubble enemy 0.1%, remaining Amount is water.PH=7.4,121 DEG C of sterilizing 30min.
Fermentative medium formula during 3, described tertiary liquid fermentation is weight ratio
(1) one-level and secondary seed tank formula
Glucose 3.30%, soybean cake powder 1.80%, dipotassium hydrogen phosphate 0.04%, yeast leaching powder 0.5%, sulphuric acid Magnesium 0.04%, peptone 0.5%, sucrose 1.60%, Semen Maydis pulp 1.10%, carbamide 0.12%, Nacl 0.22%, calcium carbonate 0.42%, bubble enemy 0.1%, surplus is water.PH=7.4,121 DEG C of sterilizing 30min.
(2) fermentation tank formula
Glucose 4.50%, soybean cake powder 2.80%, Semen Maydis powder 2.50%, peanut cake powder 0.50%, cottonseed meal 0.16%, Semen Maydis pulp 0.35, yeast leaching powder 0.75%, peptone 0.16%, Nacl 0.34, carbonic acid Calcium 0.25%, surplus are water.PH=7.0,121 DEG C of sterilizing 30min.
Described tertiary liquid fermentation comprises the following steps:
3 kinds of bottle liquid spawns of above-mentioned acquisition are carried out tertiary liquid fermentation respectively, specific as follows:
(1) prepared by first class seed pot seed
According to first class seed pot culture medium dispensing, sterilizing in first class seed pot.When tank temperature drop to 32 DEG C During left and right, culture volume is about 190L, utilize flame inoculation method access 1000ml without miscellaneous bacteria and Standard compliant bottle seed liquor.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.02MPa～ The cultivation cycle of 0.05MPa, ventilation ratio=1:0.5～1:0.6, three strain strain be respectively as follows: KY-1 12h, KY-2 13.5h、KY-3 17h。
(2) prepared by secondary seed tank seed
According to secondary seed tank culture medium dispensing, sterilizing in secondary seed tank, simultaneously by grain conductor road Sterilizing (123 DEG C of sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, Culture volume is about 2000L, by aseptic seed transferring pipeline access 190L without miscellaneous bacteria and meet The first class seed pot seed liquor of standard.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.03MPa～ The cultivation cycle of 0.05MPa, ventilation ratio=1:0.5～1:0.6, three strain strain be respectively as follows: KY-1 12h, KY-2 12.5h、KY-3 12h。
(3) prepared by fermentation tank
According to fermentation tank culture medium dispensing, sterilizing in fermentation tank, simultaneously by grain conductor road sterilizing (123 DEG C sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, medium body Long-pending be about 22000L, by aseptic seed transferring pipeline access 2000L without miscellaneous bacteria and standard compliant Secondary seed tank seed liquor.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.04MPa～0.07MPa, Ventilation ratio=1:0.8～1:1.2.Control pH=6.5-6.7 by Feeding ammonia water, added by stream simultaneously The glucose solution of 80%, controls residual sugar >=1%, until pH gos up naturally (going back up to pH >=7.5), stops Feeding ammonia water and glucose solution.When spore rate is more than 90%, terminate fermentation.Strain KY-1 ferments: Cycle=47.5h.Viable count (plate count)=105.2 hundred million/ml, residual sugar=0.43%.Put tank volume =24.3T.Strain KY-2 ferments: cycle=39h.Viable count (plate count)=101.4 hundred million/ml, Residual sugar=0.23%.Put tank volume=28.7T.Strain KY-3 ferments: cycle=60.5h.Viable count is (flat Plate counts)=125.3 hundred million/ml, residual sugar=0.22%.Put tank volume=25.4T.
4, the making of NEW TYPE OF COMPOSITE bacterial manure
(1) liquid preparation: meeting of above-mentioned fermentation gained is put three bacillus amyloliquefaciens of tank standard Fermentation liquid, is mixed in isopyknic ratio, adds mixed liquor cumulative volume about (6T) and add mixed after mixing Close the stabilizer of liquid cumulative volume 0.8%, detection viable count (plate count)=100.2 hundred million/ml.Fill, Warehouse-in.Wherein, stabilizer is sodium benzoate and aminoacid, and the mass ratio of the two is 3:2, aminoacid For lysine；
(2) prepared by powder: meeting of above-mentioned fermentation gained is put the residue three strain solution starch spore bar of tank standard The fermentation liquid of bacterium, is mixed in isopyknic ratio, suitably concentrates through falling film concentration plants, adds and concentrates The stabilizer of rear mixed liquor cumulative volume 0.8%, after adding concentration, mixed liquor cumulative volume 37% carrier is abundant Mixing (bentonite, powdered rice hulls, zeolite powder, white carbon, aminoacid, calcium carbonate), be spray-dried and ?.Detection viable count (plate count)=212.2 hundred million/ml, packaging, warehouse-in.Described carrier is swelling Soil, powdered rice hulls, zeolite powder, white carbon, aminoacid, calcium carbonate, in mass ratio 25:5:30:10: 30 ratio mixing, aminoacid is lysine；Wherein, stabilizer is sodium benzoate and aminoacid, the two Mass ratio be 3:2, aminoacid is lysine.
Embodiment 4
The incubation of three described bacillus amyloliquefaciens comprises the following steps
1, the preparation of solid slope strain
The strain of preservation is inoculated in slant medium, and in 37 DEG C of constant temperature quiescent culture 24h, microscopy is without miscellaneous bacteria And spore rate is more than 95%, and in 4 DEG C of Storage in refrigerator.Use in 1 year.
The inclined-plane formula (weight ratio) used: glucose 0.9%, yeast leaching powder 0.5%, peptone 0.5%, Carnis Bovis seu Bubali cream 0.5%, sodium chloride 0.2%, calcium chloride 0.01%, disodium hydrogen phosphate 0.04%, solubility are formed sediment Powder 2.0%, ammonium sulfate 0.01%, manganese sulfate 0.1mg/L, sodium molybdate 0.1mg/L, agar powder 1.8%, Surplus is water.PH=7.2.121 DEG C, sterilizing 30min.
2, the preparation of bottle liquid spawn
The excellent slant strains of preservation, is inoculated in liquid bottles culture medium, in 180rpm, 30 DEG C～ 32 DEG C, constant-temperature shaking culture 24h, obtain a bottle liquid spawn.
The liquid bottles formula (weight ratio) used: glucose 4.3%, soybean cake powder 3.2%, phosphoric acid hydrogen Dipotassium 0.04%, yeast leaching powder 0.5%, magnesium sulfate 0.02%, peptone 0.25%, sucrose 2.50%, Semen Maydis pulp 1.20%, carbamide 0.2%, Nacl 0.02%, calcium carbonate 0.2%, bubble enemy 0.1%, surplus For water.PH=7.4.121 DEG C of sterilizing 30min.
Fermentative medium formula during 3, described tertiary liquid fermentation is weight ratio
(1) one-level and secondary seed tank formula
Glucose 4.3%, soybean cake powder 3.2%, dipotassium hydrogen phosphate 0.04%, yeast leaching powder 0.5%, magnesium sulfate 0.02%, peptone 0.25%, sucrose 2.50%, Semen Maydis pulp 1.20%, carbamide 0.2%, Nacl 0.02%, Calcium carbonate 0.2%, bubble enemy 0.1%, surplus is water.PH=7.4.121 DEG C of sterilizing 30min.
(2) fermentation tank formula
Glucose 4.50%, soybean cake powder 3.40%, Semen Maydis powder 1.55%, peanut cake powder 1.00%, cottonseed meal 0.50%, Semen Maydis pulp 0.35%, yeast leaching powder 0.55%, peptone 0.56%, Nacl 0.34%, carbon Acid calcium 0.20%, surplus are water.PH=7.5.121 DEG C of sterilizing 30min.
Described tertiary liquid fermentation comprises the following steps
(1) prepared by first class seed pot seed
According to first class seed pot culture medium dispensing, sterilizing in first class seed pot.When tank temperature drop to 32 DEG C During left and right, culture volume is about 180L, utilize flame inoculation method access 1000ml without miscellaneous bacteria and Standard compliant bottle seed liquor.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.02MPa～ 0.05MPa, ventilation ratio=1:0.5～1:0.6.PH is natural.The cultivation cycle of three strain strains is respectively as follows: KY-1 16h、KY-2 13h、KY-3 15h。
(2) prepared by secondary seed tank seed
According to secondary seed tank culture medium dispensing, sterilizing in secondary seed tank, simultaneously by grain conductor road Sterilizing (123 DEG C of sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, Culture volume is about 1500L, by aseptic seed transferring pipeline access 180L without miscellaneous bacteria and meet The first class seed pot seed liquor of standard.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.03MPa～ 0.05MPa, ventilation ratio=1:0.5～1:0.6.PH is natural.The cultivation cycle of three strain strains is respectively as follows: KY-1 13h、KY-2 15h、KY-3 15h。
(3) prepared by fermentation tank
According to fermentation tank culture medium dispensing, sterilizing in fermentation tank, simultaneously by grain conductor road sterilizing (123 DEG C sterilizing 60min.), and use filtrated air pressurize.Time when about tank temperature drop to 32 DEG C, medium body Long-pending be about 23000L, by aseptic seed transferring pipeline access 1500L without miscellaneous bacteria and standard compliant Secondary seed tank seed liquor.Condition of culture: temperature 30 DEG C～32 DEG C, tank pressure=0.04MPa～0.07MPa, Ventilation ratio=1:0.8～1:1.2.Control pH=6.5-6.7 by Feeding ammonia water, added by stream simultaneously The glucose solution of 80%, controls residual sugar >=1%, until pH gos up naturally, stops Feeding ammonia water and Fructus Vitis viniferae Sugar juice.When spore rate is more than 90%, terminate fermentation.Strain KY-1 ferments: cycle=48h.Viable bacteria Number (plate count)=141.2 hundred million/ml, residual sugar=0.23%.Put tank volume=25.3T.Strain KY-2 sends out Ferment: cycle=53h.Viable count (plate count)=157.2 hundred million/ml, residual sugar=0.11%.Put tank volume =27.7T.Strain KY-3 ferments: cycle=46.5h.Viable count (plate count)=165.0 hundred million/ml, Residual sugar=0.22%.Put tank volume=26.4T.
4, the making of NEW TYPE OF COMPOSITE bacterial manure
(1) liquid preparation: meeting of above-mentioned fermentation gained is put three bacillus amyloliquefaciens of tank standard Fermentation liquid, is mixed in isopyknic ratio, adds mixed liquor cumulative volume about (6T) and add mixed after mixing Close the stabilizer of liquid cumulative volume 1.0%, detection viable count (plate count)=118.8 hundred million/ml.Fill, Warehouse-in.Wherein, stabilizer is sodium benzoate and aminoacid, and the mass ratio of the two is 5:1, aminoacid For lysine；
(2) prepared by powder: meeting of above-mentioned fermentation gained is put the residue three strain solution starch spore bar of tank standard The fermentation liquid of bacterium, is mixed in isopyknic ratio, suitably concentrates through falling film concentration plants, adds and concentrates The stabilizer of rear mixed liquor cumulative volume 1.0%, after adding concentration, mixed liquor cumulative volume 35% carrier is abundant Mixing (bentonite, powdered rice hulls, zeolite powder, white carbon, aminoacid, calcium carbonate), be spray-dried and ?.Detection viable count (plate count)=233.2 hundred million/ml, packaging, warehouse-in.Described carrier is swelling Soil, powdered rice hulls, zeolite powder, white carbon, aminoacid, calcium carbonate, in mass ratio 35:10:15: 20:30 ratio mixes, and aminoacid is lysine；Wherein, stabilizer is sodium benzoate and aminoacid, The mass ratio of the two is 5:1, and aminoacid is lysine.
Application examples
Above-described embodiment is utilized to obtain microbial inoculum in No. 26 canopies of Jian Fang town, Beipiao City five science and technology demonstration (98 meters) Using, crop is Fructus Lycopersici esculenti, and kind is that Europe shield is pink, uses the time: autumn stubble in 2015, whole process makes With.
Conceptual design foundation
1, soil result (TC-100 soil nutrient tacheometer) is surveyed
N(mg/kg) P2O5(mg/kg) K2O(mg/kg) Organic % pH
24.28 78.46 486.14 1% 6.5
2, Fructus Lycopersici esculenti regulation of fertilizer requirement
(1) in Fructus Lycopersici esculenti life cycle, N, P, K, Ca, Mg5 kind element absorption ratio is about (1:0.26:1.8:0.74:0.18 mass ratio)；
(2) in analysis Fructus Lycopersici esculenti whole plant body, nitrogen, phosphorus element, the mass ratio of potassium element are 1:0.4:2
(3) Fructus Lycopersici esculenti different growth stage is different to the absorbtivity of nutrient, typically increases with the prolongation of period of duration. At Seedling Stage based on nitrogen nutrition, when the first fringe fruit starts result, to nitrogen, phosphorus, the absorbtivity of potassium Increasing sharply, nitrogen accounts for 50% in three elements, and potassium only accounts for 32%；Contain the phase to result and start results Phase, nitrogen only accounts for 36%, and potassium has accounted for 50%, and the absorbtivity of fruiting period phosphorus accounts for 15%.Fructus Lycopersici esculenti needs potassium Feature be from bear fruit beginning the most linearly rise, fruit expanding period K uptake account for the time of infertility inhale More than the 70% of potassium total amount.Until gathering, the absorbtivity of potassium is the most slightly reduced by the later stage.
(4) Fructus Lycopersici esculenti nitrogen, phosphorus, the ratio of potassium dose should be 1:1:2
3, fertilizer applications: (every mu of use)
Testing program is embodied as
(1) base fertilizer/mu: silicon-calcium-magnesium 60 jin, compound fertilizer 160 jin, farmers''s excrement 5 side in above-mentioned fertilizer applications
(2) seedling processes: first carry with bacillus amyloliquefaciens composite bacteria agent capable powder 500g of the present invention before plantation Front brown sugar ferments more than 2 hours, dips in root after dilute with water 500 times, transplants seedling；
(2) field planting: 500 grams of bacillus amyloliquefaciens composite bacteria agent capable powder of the present invention and solution starch bud of the present invention The 5 kilograms of mixing of spore bacillus composite bacteria agent capable liquid preparation, ferment more than 2 hours with brown sugar the most in advance, With seedling recovering water drip irrigation.Acceleration is taken root, and controls death rate, controls intercalary growth.
(3) foliage-spray/mu
After first fringe fruit sits: use bacillus amyloliquefaciens composite bacteria agent capable liquid preparation 60ml and 90 of the present invention Jin water mixing, then foliage-spray, 7-10 days are once, until end of gathering.
(4) when watering every time, every mu with 500 grams of bacillus amyloliquefaciens composite bacteria agent capable powder of the present invention and this Invention bacillus amyloliquefaciens composite bacteria agent capable liquid preparation 5 kilograms, is spaced pouring in 10-15 days the most straight Use 6 times altogether to gathering.
4, using effect:
Effect Test group Matched group
Plant growing way Vigorous The golden mean of the Confucian school
Per plant quantity 6 layers of fruit 4 layers of fruit
Really table Light, size is uniform, mellow and full, no-dehiscent fruit Size is uneven, has corner angle, has dehiscent fruit
Average fruit weight 280 grams 220 grams
The same period price 2.0 yuan/jin 1.8 yuan/jin
The soft durometer of soil Loose, as stepping down on sponge, flexible Firmly, harden seriously
The access times of pesticide 2 times 5 times
(1) growth synchronizes, and fruit-setting rate is high: former years 4～5, basin fruit was all felt uneasy, even if fruit of bearing fruit is also few, Irregular.Within 2015, using composite bacteria agent capable on the basis of routine fertilizer, seedling sprout growing way is good, the greenest but also strong, And the comparison of maturation is concentrated, fruit becomes more in fact, and most bearing fruit is 6 basin fruits, and few is 5 basins Really, fruit is neat, and size is uniform.
(2) disease resistance: reduce the generation of pest and disease damage.The Peptic Ulcers in former years occurs the most again, agricultural use time Number reduces.
(3) improvement soil: soil property is substantially loosened, steps on sensation soil elastic
(4) yield and product quality are improved, additional income: compared output increased 30% with 2014, west Red Fructus Kaki condition is good, and major part is as border trade commodity selling, and the highest 0.2 yuan/jin of price, income ratio goes Year adds about 20000 yuan.
After being manured into soil from the above, after three bacillus amyloliquefaciens bring back to life under appropriate conditions, soon Speed breeding, decomposing organic matter provides the nutrient substance needed for plant growing, while secretion plant is thin Born of the same parents' mitogen etc., the growth of promotion root system of plant, the absorption of acceleration nutrition, the small peptide simultaneously secreted, Small molecular protein, organic acid and highly active enzyme, effectively inhibit rhizosphere pathogen and parasite Breeding, serve and take root, promote root, mulch, the effect of strong root.For crop, root good what The best
The foliage-spray of bacillus amyloliquefaciens composite bacteria agent capable liquid preparation of the present invention so that sarcocarp and peel Cell constant speed divides, and swollen fruit is fast, and dehiscent fruit significantly reduces.Improve mouthfeel, sweet and sour taste simultaneously.No Malformed fruit occurs.
Beneficial microorganism adds, and improves crumb structure and the microecological environment of soil, water conservation fertilizer conservation, Fertilizing soil soil fertility, advantage has been built in the growth for crop.
Application examples 2
Utilize above-described embodiment to obtain microbial inoculum to use in the booth of remote modern agriculture base 20, Lingyuan, Liaoning east, Crop is Capsicum annuum L. (Fructus Capsici), uses the time: JIUYUE in 2015 16 days～on April 30th, 2016.
Fertilising foundation:
1, soil result (TC-100 soil nutrient tacheometer) is surveyed
Comparison 21# canopy
N(mg/kg) P2O5(mg/kg) K2O(mg/kg) Organic % pH
23.36 138.10 530.67 1～2 6.5
Test 20# canopy
2, the regulation of fertilizer requirement of Capsicum annuum L. (Fructus Capsici)
(1) compared with Fructus Lycopersici esculenti, Fructus Solani melongenae, the uptake characters of Fructus Capsici is much like with them, is also that K uptake is the highest, Next to that nitrogen, phosphorus is minimum.Fructus Capsici belongs to high nitrogen, middle phosphorus, high potassium type in vegetable, needs fertile total amount relatively Many.
(2) trophophase of Fructus Capsici is long, but root system is undeveloped, and root amount is few, buries shallow, the most drought-enduring also intolerant to waterlogging. Its fertilizer requirement is more than Fructus Lycopersici esculenti and Fructus Solani melongenae, and resistance to fertile ability is strong.Often produce 1 ton of Fructus Capsici, its nutrient Absorbtivity is N4.91 kilogram, P2O51.19 kilograms, K2O6.02 kilogram, its assimilation ratio is 1:0.2:1.2
(3) the calcium nutrition of Fructus Capsici the most should not be ignored, and once calcium deficiency easily makes fruit generation blossom-end rot, thus leads Cause fruit quality is substantially reduced
Fertilizer applications/mu
Testing program is embodied as
1, base fertilizer: silicon-calcium-magnesium 30 kgs/acre and compound fertilizer's 150kg/ mu in above-mentioned fertilizer applications.
Using method: above fertilizer is uniformly sprinkled into soil, ploughs deeply, rotary tillage, ridging, fill foot pre-sowing water, Wait to plant.(after being spaced 3-5 days, plant Seedling.)
2, field planting: with 500 grams/acre of bacillus amyloliquefaciens composite bacteria agent capable powder of the present invention and Xie Dian of the present invention Afnyloliquefaciens composite bacteria agent capable liquid preparation 5 kgs/acre, in advance fermentation more than 3 hours, with water by 500 Times dissolved dilution, executes or drip irrigation with seedling recovering water punching.Acceleration is taken root, and controls death rate, controls internode raw Long.
3, after the first fringe fruit sits: foliage-spray: bacillus amyloliquefaciens composite bacteria agent capable liquid preparation of the present invention Dilute with water 600 times, foliage-spray, 10 days are once, until before end of gathering.
When 4, watering, with bacillus amyloliquefaciens composite bacteria agent capable liquid preparation of the present invention 5 kgs/acre every time, Punching is executed or drip irrigation, the bacillus amyloliquefaciens minimum use of composite bacteria agent capable liquid preparation of the present invention 3 times.
5, other booths are by the remedial after freeze injury: each booth of being injured, and use solution starch spore of the present invention Bacillus composite bacteria agent capable powder and bacillus amyloliquefaciens composite bacteria agent capable liquid preparation of the present invention mixing, then Carry out 400 times of dilutions with water, combine punching and execute, be spaced 7 days once, continuous 3 times.
Use background:
In January, 2016, when ground temperature 4 DEG C on the low side compared with the same period in former years～5 DEG C, outdoor reaches subzero 23 DEG C of .2016 In morning about 9 on January 20, garden has a power failure suddenly more than 6 hours, protects canopy curtain and cannot put down. The low temperature of more than ten day, adds power failure impact, causes freeze injury large area to occur, nearly 80 of matched group The Capsicum annuum L. rice shoot of booth has not had growing point, spends few.The underproduction about 2/3rds compared with the same period of last year.Every day Shipment only has about 20 tons.And the Capsicum annuum L. rice shoot testing process group is not affected by a bit freeze injury, Growing way is vigorous, achieves great success.Then we are by same processing method, to the matched group that freeze injury occurs 80 booths remedy, after 10 days, growing point breaks up again, yields positive results.Compensate for loss.
Using effect:
The actual application of the solution starch composite bacteria agent capable of the present invention that above-described embodiment obtains, fully demonstrates it degeneration-resistant Property, especially winter resistance.
The general performance of test No. 20 booths of canopy:
1, after transplanting, seedling-slowing stage is short, takes root fast, and survival rate is high.
2, trophophase branch is the most full of leaves, it is possible to freeze proof evil, anti-continuous cropping, suppresses bacteriosis.
3, growth cycle is long, spends even fruit many.
4, fruit is big uniformly, commodity rate is high, improves yield, increases benefit.
The bacillus amyloliquefaciens composite bacteria agent capable using the invention described above acquisition in sum has a characteristic that
1, base manure: Fructus Capsici is long due to trophophase, root system is more weak, in order to promote in time after field planting growth of seedling and Later stage plant bears fruit more, use bacillus amyloliquefaciens composite bacteria agent capable liquid preparation of the present invention powder and 500 times of water diluents of liquid preparation dip in root, use as seedling recovering water again after field planting.One, accelerate The breeding of bacillus amyloliquefaciens viable bacteria, two, the metabolite in compound bacteria solid powder can kill The pathogen that dead seedling root carries, the plant immunization renovation agent in liquid preparation, can repair and divide Root cell injured during Seedling, makes injured wound surface quickly-healing, prevents the intrusion of pathogen. Serve and take root, promote root, mulch, the effect of strong root, so, the survival rate of rice shoot significantly improves.
2, topdress: " upper spray " three strain composite bacteria liquid body preparations；" lower filling " bacillus amyloliquefaciens of the present invention is multiple The powder of conjunction microbial inoculum liquid preparation and 400 times of water diluents of liquid preparation, minimum more than 3 times.Three The viable bacteria of the functional bacillus amyloliquefaciens of strain, runs into suitable condition and breeds rapidly, produce antibacterial killing The somatotrophic metabolite of worm, quickly reduces the harmful microorganism in root agent soil, promotes root system development, Induction crop produces resistance, so, trophophase branch is the most full of leaves, it is possible to freeze proof evil, anti-continuous cropping, presses down Bacteriosis processed.This is also the principle place of " remedial ".
Application examples 3
Utilizing the microbial inoculum that above-described embodiment obtains, use in new people's Liuhe ditch apple orchard, Liaoning, crop is cold richness Fructus Mali pumilae, apple tree present situation: the age of tree 6 years, the fruit of 2015 bagging, there is part fruit tree to occur in that root Maize ear rot, leaf rolling.10 trees that root rot is serious are at death's door, and main the having decided in garden abandons controlling Treat.
One, for the therapeutic scheme of 10 sick trees:
1, radical cure root rot
(1) debridement focus: peel the bark of rotten place off, digs up " xylem " that variable color is downright bad.
(2) sick tree processes: by bacillus amyloliquefaciens composite bacteria agent capable liquid preparation of the present invention through water-reducible 300 Times diluent, takes the deep soil (less than 30 centimetres, as far as possible reduce the quantity of pathogen) away from lesion, With become mud, be bonded at the affected part of debridement, then snarl with the cloth of clean good air permeability, Wait healing.
(3) powder and the mixing of liquid preparation of bacillus amyloliquefaciens composite bacteria agent capable of the present invention simultaneously, are utilized Thing carries out pouring root through water-reducible 400 times of diluents.Continuous 2 times, it is spaced 7-10 days
Two, for the fruit tree of normal growth: promote the scheme of fruit quality
Of the present invention bacillus amyloliquefaciens composite bacteria agent capable liquid preparation is used in combination in fruit expanding period and coloring phase Through water-reducible 300 times of dilutions, spray in conjunction with conventional use of foliage-spray fertilizer.Interval 7-10 My god, spray 2-3 time continuously
Three, using effect:
1, come back to life as 10 the apple tree mysteries being at death's door.The most again take root foliation, and Growing way is fine, yields positive results.
2, other normal fruit trees, under technical staff instructs, are used in combination in fruit expanding period and coloring phase Solution starch composite bacteria agent capable liquid preparation of the present invention is through water-reducible 300 times of dilutions, in conjunction with conventional use of Foliage-spray fertilizer sprays.Fruit is big uniformly, and color and luster is consistent, and really table is vivid, and mouthfeel is crisp sweet, anti- Oxidation, shelf-stable, by the consistent favorable comment of client, fall over each other in eagerness to buy.
Use and as 10 the apple tree mysteries being at death's door after above-described embodiment obtains composite bacteria agent capable, play dead returning Raw, specific as follows:
(1) root rot pathogeny: this disease is the most similar to macerating root symptom, belongs to fungal disease.Pathogenic bacteria is at soil Neutralize and pass the winter on invalid body, general many in the morbidity of late March to early April, enter the morbidity Sheng phase in May. The rhizome of morbidity:
(2) the three functional bacillus amyloliquefaciens of strain contained in bacillus amyloliquefaciens composite bacteria agent capable of the present invention Living bacterial liquid body preparation, there is the function quickly repairing damaged cell, the affected area after debridement can be made impaired Plant cell recover normal physiological function；The metabolism of three bacillus amyloliquefaciens additionally contained Product, effectively inhibits the intrusion of pathogen, and the growth and breeding for regenerative cell creates safe ring Border.
(3) " take stopgap measures " and more " to effect a permanent cure ": root rot is really the soil-borne disease of fungoid, owing to root is rotten Rotten, the function absorbing moisture and nutrient gradually weakens, and last Herb is dead.Mainly show as whole strain leaf Sheet turns to be yellow, withers.So, only eradicate the pathogen of " root rot ", could fundamentally stop The generation of root rot.The powder and the liquid preparation that utilize solution starch composite bacteria agent capable liquid preparation of the present invention mix Close, carry out pouring root through water-reducible 400 times of diluents.Continuous 2 times, it is spaced 7-10 days.Wherein The spore of the bacillus amyloliquefaciens of useful work, in soil, runs into the condition (temperature of suitable sprouting Degree, humidity, air, organic matter) substantial amounts of Fast-propagation, secretion suppression is bacillary and fungoid Metabolite (small peptide, small protein, organic acid, high activity enzyme etc.), has in this, as it The weapon of profit carries out " life-and-death struggle " with the pathogen of soil-borne disease, and the growth for crop root creates Favorable environment.The deterioration that suppression root system rots.
And of the present invention bacillus amyloliquefaciens composite bacteria agent capable liquid is used in combination in fruit expanding period and coloring phase Preparation, through water-reducible 300 times of diluents, sprays in conjunction with conventional foliage-spray fertilizer.Interval 7-10 My god, spray 2-3 time continuously.Promote fruit quality.
Above-described embodiment is used to obtain composite bacteria agent capable, because it contains abundant high activity metabolite, it is possible to protect Flower and fruit, improves fruit-setting rate；Sarcocarp and the division of pericarp membrane constant speed can be accelerated, coordinate foliage-spray, Quickly absorb trace element, improve the utilization rate of fertilizer, and then promote fruit quality, there is list Really weight increase, lovely luster, antioxidation, improve effect of mouthfeel.

Claims (10)

1. a bacillus amyloliquefaciens, it is characterised in that: bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) CGMCCNO.12306 (KY-1), CGMCCNO.12307 it are respectively as follows: (KY-2), CGMCCNO.12308 (KY-3), be all preserved in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date It it is on March 24th, 2016.
2. a composite bacteria agent capable, it is characterised in that: bacillus amyloliquefaciens described in claim 1 is divided Not through tertiary liquid fermentation, the most by volume (5～1): (1～5): (5～1) ratio will fermentation Liquid mixing i.e. obtains composite bacteria agent capable.
3. the composite bacteria agent capable as described in claim 2, it is characterised in that: above-mentioned in described composite bacteria agent capable Spore rate >=90% in bacterial strain fermentation liquid after tertiary liquid fermentation respectively, blood cell count plate >=100 Hundred million/ml, residual glucose≤0.6%, pH=7.0～8.0.
4. the composite bacteria agent capable as described in claim 2, it is characterised in that: by mix according to the above ratio Mixed fermentation liquid adds mixed fermentation liquid and amasss the stabilizer of 0.1～2.0%, i.e. obtain liquid preparation.
5. the composite bacteria agent capable as described in claim 2, it is characterised in that: by mix according to the above ratio The 1/2-1/3 that mixed fermentation liquid amasss through falling film concentration to mixed fermentation liquid, then adds and mixes after concentrating Fermentating liquid volume 0.1～the stabilizer of 2.0% and after concentrating mixed fermentation liquid amass the carrier of 30%～40%, It is sufficiently mixed after addition, is spray-dried after mixing, obtains compound bacteria powder.
6. the composite bacteria agent capable as described in claim 4 or 5, it is characterised in that: described stabilizer is benzene Sodium formate and/or aminoacid；Carrier is bentonite, powdered rice hulls, zeolite powder, white carbon and calcium carbonate； Wherein, aminoacid is lysine.
7. the preparation method of the composite bacteria agent capable described in a claim 2, it is characterised in that:
The liquid seeds liquid of 3 kinds of bacillus amyloliquefaciens is seeded to first order seed by 0.5%～1% respectively In tank culture medium, in 30 DEG C～32 DEG C, tank pressure=0.02MPa～0.05MPa, ventilation ratio=1:0.5～1: 0.8, carry out one grade fermemtation under first class seed pot cycle=12h～24h and cultivate to Biomass >=10%, thalline Form is consistent, and rod-short, two ends are concordant, colour homogeneous, and pH gos up naturally to 6.5～7.0, stand-by；
Above-mentioned one grade fermemtation culture fluid is seeded in secondary seed tank culture medium by 1-15%, in 30 DEG C～ 32 DEG C, tank pressure=0.02MPa～0.05MPa, ventilation ratio=1:0.5～1:0.8, secondary seed tank is trained Carrying out second order fermentation under foster cycle=12h～24h to cultivate to Biomass >=10%, thalli morphology is consistent, short Shaft-like, two ends are concordant, colour homogeneous, and pH gos up naturally to 6.5～7.0, stand-by；
Above-mentioned second order fermentation culture fluid 2-15% is seeded in fermentation tank culture medium, in 30 DEG C～32 DEG C , tank pressure=0.04MPa～0.07MPa, ventilation ratio=1:0.8～1:1.2, pH=6.5-6.7；Treat pH Naturally go up, The glucose solution that pH=7.0～8.0 streams add 80%, cycle=30h～72h, obtain three grades the most respectively and send out Ferment liquid；The glucose solution of 80% is prepared and in 115 DEG C, 20 minutes sterilizations, fall in joining sugar bowl After warm to 30 DEG C, addition is 10L～15L per minute, then according to volume ratio (5～1): (1～5 ): fermentation liquid mixing is i.e. obtained composite bacteria agent capable by (5～1) ratio.
8. the preparation method of the composite bacteria agent capable as described in claim 7, it is characterised in that: will be by above-mentioned The mixed fermentation liquid of ratio mixing adds mixed fermentation liquid and amasss the stabilizer of 0.1-2.0%, i.e. obtain Liquid preparation.
9. the preparation method of the composite bacteria agent capable as described in claim 7, it is characterised in that: will be by above-mentioned The mixed fermentation liquid of ratio mixing, through the 1/2 1/3 of falling film concentration to mixed volume, then adds concentration The stabilizer of rear volume 0.1-2%, after adding concentration, the carrier of volume 30%～40% is sufficiently mixed, spray Mist is dried and obtains compound bacteria powder.
10. the preparation method of the composite bacteria agent capable as described in claim 8 or 9, it is characterised in that: institute Stating stabilizer is sodium benzoate and/or aminoacid；Carrier is bentonite, powdered rice hulls, zeolite powder, Linesless charcoal Black and calcium carbonate；Wherein, aminoacid is lysine.
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