CN105439657B - A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry - Google Patents
A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry Download PDFInfo
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- CN105439657B CN105439657B CN201510948301.1A CN201510948301A CN105439657B CN 105439657 B CN105439657 B CN 105439657B CN 201510948301 A CN201510948301 A CN 201510948301A CN 105439657 B CN105439657 B CN 105439657B
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/50—Treatments combining two or more different biological or biochemical treatments, e.g. anaerobic and aerobic treatment or vermicomposting and aerobic treatment
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention belongs to technical field of biological organic fertilizer, to solve the problems, such as the prevention of disease in replanting and providing strawberry biological organic fertilizer simultaneously, provide a kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry.Bacteria residue mixing after animal dung and edible fungus culturing is the special biologic organic fertilizer of resisting repeated stubbles of strawberry with the mixture of bacteria residue and animal dung after bacillus amyloliquefaciens JN2, Rhodopseudomonas palustris and fermentation of Aspergillus niger edible fungus culturing.JN2 has stronger antagonism to the main pathogenic fungi of disease in replanting, has good cellulose degradation to act on, and Rhodopseudomonas palustris enhances plant nutrient and absorbs, detoxifies and stimulate plant growth；Aspergillus niger decomposition of cellulose.There is higher preventive effect to disease in replanting.Have the characteristics that nutrient diversity, harmony and long-term effect, colonizes and be proliferated conducive to flora.The biological organic fertilizer is that medicine is also fertilizer, solves the problems, such as the disease of replanted strawberry and fertility, and super quality and competitive price have good application prospect and Development volue.
Description
Technical field
The invention belongs to technical field of biological organic fertilizer, and in particular to a kind of system of the special biologic organic fertilizer of resisting repeated stubbles of strawberry
Preparation Method.
Background technology
" strawberry " is known as the title of " fruit queen ", is loved by consumers.But " continuous cropping disease " is a disaster of cultivated strawberry
The problems such as topic, the strawberry shoot survival percent that brings is low, result is few, the abnormal fruits of Bing Guo are more, nutrient quality decline to strawberry cultivating person with
Great economic loss is carried out.Many researchs point out, the Etiological of " continuous cropping disease ", which is attributed to soil surface characters, to be increased, from poisonous substance
Matter increase with soil nutrient elements wane three aspect.Advocating green, environmentally friendly, organic, No-harmful apple orchard today, chemical pesticide
Just gradually replaced by biological pesticide, the usage amount of chemical fertilizer is also being reduced, and the importance of organic fertilizer is mentioned new height.
Mushroom dreg is to be cultivated the later Waste compost of bacterium, is often made of straw, corncob, wheat straw etc., after cultivation
Bacteria residue not only contains all kinds of nutriments such as crude protein, crude fibre, crude fat, nitrogen, phosphorus, potassium, calcium, amino acid, also contains type
Various, substantial amounts microbiologic populations, especially cellulose degradation class fungi are more, the cellulose degradation that they are secreted
Enzyme and hydrolase are also more.A large amount of bacteria residues can be all generated in the place of cultivated mushroom every year, these bacteria residues are often thrown with regard to mushroom grower
It abandons, this is not only the waste to resource, also can cause environmental pollution.The Fertilizer Transformed of mushroom slag is always that mushroom slag effectively recycles
One of important channel.Such as patent（Application number：200910262948.3）It is exactly that microbial fermentation is used to mushroom slag and fowl and animal excrement
Technology recycles after being handled as artificial matrices or organic fertilizer.Mushroom slag super quality and competitive price, as fermentation substrate and function
Strain, which matches, is made bio-bacterial manure, there is good application prospect and Development volue.
Application No. is：201210021687.8 entitled：《It is a kind of prevention disease in replanting biological prevention and control agent and preparation side
Method》Patent of invention in mention it is a kind of prevention disease in replanting biological prevention and control agent, with hundred mixture of mountain（The weight such as kaempferia galanga and the tuber of stemona
Than）, corn stalk powder, wheat bran be solid material, be that biocontrol microorganisms are made biological prevention and control agent and are well suited for for strawberry continuous cropping to solve phenol bacterium
Plot, after said preparation applies continuous cropping plot, the occurring degree of the substantially reduced soil-borne disease of energy reduces strong suppression harmful bacteria pair in soil
The content of hydroxybenzoic acid and benzoic acid promotes the development of strawberry growth indexes, can finally significantly improve yield of strawberry.The present invention
Product is primarily adapted for use in the prevention of the soil-borne diseases such as strawberry droop, sclerotiniose and verticillium wilt, is conducive to optimize strawberry continuous cropping soil
The physicochemical character of earth, it is free from environmental pollution, it is at low cost to safety of human and livestock, using convenient.But contain kuh-seng, trifoliate orange in microbial inoculum formula
Real equal 6 kinds of Chinese medicines, source is not very extensive, and what he obtained is bacteria agent finished product, therefore makes and be not easy, and is difficult to
It is enlarged metaplasia production, and the effect of only biological pesticide is without the effect of bio-feritlizer.
Invention content
The present invention provides one to solve the problems, such as the prevention of disease in replanting and provide strawberry biological organic fertilizer simultaneously
The preparation method of the kind special biologic organic fertilizer of resisting repeated stubbles of strawberry.
The present invention is realized by following technical solution：A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry, will
After animal dung and edible fungus culturing bacteria residue mixing, with bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
JN2, Rhodopseudomonas palustris（Rhodop seudanonas palustris）With aspergillus niger (AspergillusNige it) sends out
The mixture of bacteria residue and animal dung after ferment edible fungus culturing, the as special biologic organic fertilizer of resisting repeated stubbles of strawberry, specific preparation side
Method is as follows：
（1）Matrix mixes：By the bacteria residue after edible fungus culturing with cow dung by weight 1:1 is uniformly mixed, and adjusts C/N ratios
It is 20 ~ 30, pH value is adjusted to neutrality, moisture regulation to 55-65%；
（2）The preparation of microbial inoculum：
A. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 bacterium solutions preparation：The solution of 4 DEG C of preservations
Bacillus amyloliquefaciens JN2 is inoculated on sodium carboxymethylcellulose fluid nutrient medium 37 DEG C, and 100 ~ 150rpm shaking table cultures are stayed overnight, system
For at seed liquor；
By seed liquor by volume 1% ~ 5% inoculum concentration be inoculated in the sodium carboxymethylcellulose liquid to sterilize in fermentation tank
In culture medium, tank internal pressure keeps 0.02-0.04MPa, 37 DEG C, 1.8 ± 0.2mg/L of dissolved oxygen, rotating speed 150r/min of temperature, fermentation
Culture two days obtains JN2 zymotic fluids, and it is 4 × 10 to detect living bacteria count in zymotic fluid8cfu/mL；
B. Rhodopseudomonas palustris（Rhodop seudanonas palustris）The preparation of bacterium solution：GH culture mediums are filled
Rhodopseudomonas palustris strain aseptic condition is inoculated in GH culture mediums by whole branch test tube, 30 DEG C of temperature, intensity of illumination 5000Lx
Anaerobic culturel is stood, until bacterium solution becomes peony up to seed liquor；
Rhodopseudomonas palustris is expanded and is cultivated, culture medium and condition of culture are identical as the method for seed liquor is prepared；Work as bacterium
When liquid color becomes laking, obtain Rhodopseudomonas palustris zymotic fluid, detect living bacteria count in zymotic fluid be 4 ×
108cfu/mL；
C. aspergillus niger (AspergillusNige) the preparation of microbial inoculum：It is solid that aspergillus niger strain is inoculated in potato glucose
On body culture medium flat plate, 28 DEG C of temperature are inverted dark culturing and choose mycelium inoculation in potato glucose after growing fresh mycelia
In fluid nutrient medium, 28 DEG C, 100 ~ 150rpm shaking table cultures obtain aspergillus niger after growing a large amount of hypha bodies in culture medium
Seed liquor；
Wheat bran watering is mixed thoroughly, is made the bran mass of moisture content 55-65%, it is cold after 110-120 DEG C of high pressure sterilization
But, the ratio for 150mL bacterium solutions being accessed in every kilogram of bran mass accesses aspergillus niger seed liquor, stirs evenly, and 28 DEG C stand training
It supports to a large amount of spores and stops culture when generating, culture is placed in after 30 DEG C of drying in oven up to aspergillus niger bacterium powder；
（3）Microbe inoculation ferments：It is added into mixed fertilizer in the ratio for accessing 10kg in every square metre of mixed fertilizer
Aspergillus niger bacterium powder banks up fermentation after mixing thoroughly, continue to ferment after turning when temperature is less than 50 DEG C, and fermentation time is 30 ~ 45 days；Hair
After the completion of ferment, the red vacation of bacillus amyloliquefaciens bacterium solution and marsh is separately added into the ratio for accessing 10L in every square of mixed fertilizer
Monad bacterium solution, adjustment pH value is 6 ~ 7, moisture 55-65%, and after stirring evenly, booth carries out the at the thin layer of 15-25cm
Secondary fermentation, controlled at 25 ~ 30 DEG C, after fermentation 7 ~ 10 days, be stirred for uniformly air-drying to moisture content≤15% to get to
The special biologic organic fertilizer of resisting repeated stubbles of strawberry.
The sodium carboxymethylcellulose culture medium prescription is：Sodium carboxymethylcellulose 15g/L, yeast powder 1g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 1g/L, ammonium nitrate 1g/L, magnesium sulfate 0.5g/L；The GH culture medium prescriptions are：Potassium dihydrogen phosphate 1g/L, sodium acetate 1g/
L, magnesium sulfate 0.5g/L, ammonium sulfate 1g/L, sodium chloride 1g/L, calcium chloride 0.1g/L, sodium succinate 1g/L, yeast powder 0.5g/L,
Peptone 0.5g/L, pH value 6.2.
The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 is in Chinese microorganism strain preservation
Administration committee's common micro-organisms center preservation, deposit number are：CGMCC No:10677, the deposit date is：April 1 in 2015
Day, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode
It is 100101.
The Rhodopseudomonas palustris（Rhodop seudanonas palustris）Strain is purchased from Chinese agriculture microorganism
Culture presevation administrative center, number ACCC10649.The aspergillus niger (AspergillusNige) strain is common purchased from China
Microbiological Culture Collection administrative center, number CGMCC3.6477.
Total living bacteria count content >=4 × 10 of the special biologic organic fertilizer of resisting repeated stubbles of prepared strawberry8CFU/mL。
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) JN2 for strawberry weight
The main pathogenic fungi of stubble disease, to Botrytis cinerea（Botrytis cinereaPers., cause gray mold）, Fusarium
（FusariumSpp., cause droop）, quasi- Pestalotia（Pestalotiopsis, cause leaf spot）With relatively strong short of money
Anti- effect, while having good cellulose degradation effect concurrently, with Rhodopseudomonas palustris（Rhodop seudanonas
palustris）With aspergillus niger (AspergillusNige) compound to be used as functional flora.Rhodopseudomonas palustris has enhancing
Plant nutrient absorbs, detoxifies and stimulates plant growth effect；Aspergillus niger has stronger capacity of decomposition to cellulose.It will be cheap
And bacteria residue after the extensive edible fungus culturing of resource and animal dung be as primary raw material, with the complex function microflora fermentation of preparation,
Product after fermentation is that can effectively prevent the biological organic fertilizer that strawberry continuous cropping is hindered.
Difference lies in it is same with other biological organic fertilizers for the special biologic organic fertilizer of resisting repeated stubbles of strawberry prepared by the present invention
When have the function of following two kinds：（1）It has stronger antagonism for main three kinds of pathogens of disease in replanting, to strawberry
Continuous cropping disease has higher preventive effect.（2）In bacteria residue after edible fungus culturing containing abundant organic matter, mineral element, trace element,
Organic nutrient substance and bioactive ingredients etc., on the one hand so that the organic fertilizer produced as primary raw material using it has nutrient
The characteristics of diversity, harmony and long-term effect, another side provide preferable substance for the functional microorganism strain growth of addition
Basis is conducive to colonizing and being proliferated for functional flora.The biological organic fertilizer is that medicine is also fertilizer, and one recruits the disease for solving replanted strawberry
Evil and fertility problem, super quality and competitive price, there is good application prospect and Development volue.
Description of the drawings
Fig. 1 be bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 is solid in sodium carboxymethylcellulose
Congo red staining shows the result figure of transparent circle after being cultivated 2 days on body tablet；Fig. 2 is that JN2 bacterial strains are solid in sodium carboxymethylcellulose
Morphological feature figure on body tablet；Fig. 3 is that JN2 strains observe cellular morphology figure under 40 power microscopes；Fig. 4 is living tissue method
Preventive effect experimental result picture of the JN2 bacterial strains to gray mold is measured, left figure is the sterile water process group of control group in figure, and right figure is sent out for JN2
Zymotic fluid processing group；Fig. 5 is tablet face-off knots of the bacillus amyloliquefaciens JN2 to Fusarium and quasi- Pestalotia pathogen
Fruit is schemed, in figure：A：Sickle-like bacteria compares；B sickle-like bacteria stand facing each other with JN2；C：Quasi- pestalotia bacteria control；D：Quasi- pestalotia bacteria with
JN2 stands facing each other；Fig. 6 is the Glucose standards of filter paper enzyme activity and carboxymethylcelluloenzyme enzyme activity that reducing sugar method measures JN2 bacterial strains
Curve graph；Fig. 7 is the cellulase activity testing result figure to JN2 bacterial strains under different substrate for induction；Fig. 8 is different fermentations temperature pair
The cellulase activity testing result figure of JN2 bacterial strains.
Specific implementation mode
The present invention is further elaborated with reference to embodiments, but is not used to limit the present invention.
Embodiment 1：A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry, after animal dung and edible fungus culturing
Bacteria residue mixing, with bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2, Rhodopseudomonas palustris
（Rhodop seudanonas palustris）With aspergillus niger (AspergillusNige) the bacterium after fermentation edible fungus culturing
The special biologic organic fertilizer of resisting repeated stubbles of the mixture of slag and animal dung, as strawberry, specific preparation method are as follows：
（1）Matrix mixes：By the bacteria residue after edible fungus culturing with cow dung by weight 1:1 is uniformly mixed, and adjusts C/N ratios
It is 20, pH value is adjusted to neutrality, moisture regulation to 55%；
（2）The preparation of microbial inoculum：
A. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 bacterium solutions preparation：The solution of 4 DEG C of preservations
Bacillus amyloliquefaciens JN2 is inoculated on sodium carboxymethylcellulose fluid nutrient medium 37 DEG C, and 100rpm shaking table cultures are stayed overnight, and are prepared into
Seed liquor；
By seed liquor by volume 1% inoculum concentration be inoculated in the sodium carboxymethylcellulose Liquid Culture to sterilize in fermentation tank
In base, tank internal pressure keeps 0.02MPa, 37 DEG C, dissolved oxygen 2.0mg/L, rotating speed 150r/min of temperature, fermented and cultured two days to obtain
JN2 zymotic fluids, it is 4 × 10 to detect living bacteria count in zymotic fluid8cfu/mL；
B. Rhodopseudomonas palustris（Rhodop seudanonas palustris）The preparation of bacterium solution：GH culture mediums are filled
Rhodopseudomonas palustris strain aseptic condition is inoculated in GH culture mediums by whole branch test tube, 30 DEG C of temperature, intensity of illumination 5000Lx
Anaerobic culturel is stood, until bacterium solution becomes peony up to seed liquor；
Rhodopseudomonas palustris is expanded and is cultivated, culture medium and condition of culture are identical as the method for seed liquor is prepared；Work as bacterium
When liquid color becomes laking, obtain Rhodopseudomonas palustris zymotic fluid, detect living bacteria count in zymotic fluid be 4 ×
108cfu/mL；
C. aspergillus niger (AspergillusNige) the preparation of microbial inoculum：Aspergillus niger strain is inoculated in conventional PDA solids training
It supports on base tablet, 28 DEG C of temperature are inverted dark culturing and choose mycelium inoculation in potato glucose liquid after growing fresh mycelia
In culture medium, 28 DEG C, 100rpm shaking table cultures obtain aspergillus niger seed liquor after growing a large amount of hypha bodies in culture medium；
Wheat bran watering is mixed thoroughly, is made the bran mass of moisture content 55%, 110-120 DEG C of high pressure sterilization postcooling,
The ratio that 150mL bacterium solutions are accessed in every kilogram of bran mass accesses aspergillus niger seed liquor, stirs evenly, 28 DEG C of stationary cultures are extremely
A large amount of spores stop culture when generating, and culture is placed in after 30 DEG C of drying in oven up to aspergillus niger bacterium powder；
（3）Microbe inoculation ferments：It is added into mixed fertilizer in the ratio for accessing 10kg in every square metre of mixed fertilizer
Aspergillus niger bacterium powder banks up fermentation after mixing thoroughly, continue to ferment after turning when temperature is less than 50 DEG C, and fermentation time is 30 days；Fermentation
After the completion, it is separately added into bacillus amyloliquefaciens bacterium solution in the ratio for accessing 10L in every square of mixed fertilizer and marsh is red false single
Born of the same parents' bacterium bacterium solution, adjustment pH value are 6, moisture 55%, and after stirring evenly, booth carries out second at the thin layer of 15cm and ferments, control
Temperature processed is 25 DEG C, after fermenting 10 days, is stirred for uniformly air-drying to moisture content≤15% and be given birth to get to the special anti-continuous cropping of strawberry
Organic fertilizer.
The sodium carboxymethylcellulose culture medium prescription is：Sodium carboxymethylcellulose 15g/L, yeast powder 1g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 1g/L, ammonium nitrate 1g/L, magnesium sulfate 0.5g/L；The GH culture medium prescriptions are：Potassium dihydrogen phosphate 1g/L, sodium acetate 1g/
L, magnesium sulfate 0.5g/L, ammonium sulfate 1g/L, sodium chloride 1g/L, calcium chloride 0.1g/L, sodium succinate 1g/L, yeast powder 0.5g/L,
Peptone 0.5g/L, pH value 6.2.
The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 is in Chinese microorganism strain preservation
Administration committee's common micro-organisms center preservation, deposit number are：CGMCC No:10677, the deposit date is：April 1 in 2015
Day, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode
It is 100101.Total living bacteria count content >=4 × 10 of the special biologic organic fertilizer of resisting repeated stubbles of prepared strawberry8CFU/mL。
Embodiment 2：A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry, after animal dung and edible fungus culturing
Bacteria residue mixing, with bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2, Rhodopseudomonas palustris
（Rhodop seudanonas palustris）With aspergillus niger (AspergillusNige) the bacterium after fermentation edible fungus culturing
The special biologic organic fertilizer of resisting repeated stubbles of the mixture of slag and animal dung, as strawberry, specific preparation method are as follows：
（1）Matrix mixes：By the bacteria residue after edible fungus culturing with cow dung by weight 1:1 is uniformly mixed, and adjusts C/N ratios
It is 25, pH value is adjusted to neutrality, moisture regulation to 60%；
（2）The preparation of microbial inoculum：
A. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 bacterium solutions preparation：The solution of 4 DEG C of preservations
Bacillus amyloliquefaciens JN2 is inoculated on sodium carboxymethylcellulose fluid nutrient medium 37 DEG C, and 120rpm shaking table cultures are stayed overnight, and are prepared into
Seed liquor；
By seed liquor by volume 3% inoculum concentration be inoculated in the sodium carboxymethylcellulose Liquid Culture to sterilize in fermentation tank
In base, tank internal pressure keeps 0.03MPa, 37 DEG C, dissolved oxygen 1.6mg/L, rotating speed 150r/min of temperature, fermented and cultured two days to obtain
JN2 zymotic fluids, it is 4 × 10 to detect living bacteria count in zymotic fluid8cfu/mL；
B. Rhodopseudomonas palustris（Rhodop seudanonas palustris）The preparation of bacterium solution：GH culture mediums are filled
Rhodopseudomonas palustris strain aseptic condition is inoculated in GH culture mediums by whole branch test tube, 30 DEG C of temperature, intensity of illumination 5000Lx
Anaerobic culturel is stood, until bacterium solution becomes peony up to seed liquor；
Rhodopseudomonas palustris is expanded and is cultivated, culture medium and condition of culture are identical as the method for seed liquor is prepared；Work as bacterium
When liquid color becomes laking, obtain Rhodopseudomonas palustris zymotic fluid, detect living bacteria count in zymotic fluid be 4 ×
108cfu/mL；
C. aspergillus niger (AspergillusNige) the preparation of microbial inoculum：Aspergillus niger strain is inoculated in conventional PDA solids training
It supports on base tablet, 28 DEG C of temperature are inverted dark culturing and choose mycelium inoculation in potato glucose liquid after growing fresh mycelia
In culture medium, 28 DEG C, 120rpm shaking table cultures obtain aspergillus niger seed liquor after growing a large amount of hypha bodies in culture medium；
Wheat bran watering is mixed thoroughly, is made the bran mass of moisture content 60%, 110-120 DEG C of high pressure sterilization postcooling,
The ratio that 150mL bacterium solutions are accessed in every kilogram of bran mass accesses aspergillus niger seed liquor, stirs evenly, 28 DEG C of stationary cultures are extremely
A large amount of spores stop culture when generating, and culture is placed in after 30 DEG C of drying in oven up to aspergillus niger bacterium powder；
（3）Microbe inoculation ferments：It is added into mixed fertilizer in the ratio for accessing 10kg in every square metre of mixed fertilizer
Aspergillus niger bacterium powder banks up fermentation after mixing thoroughly, continue to ferment after turning when temperature is less than 50 DEG C, and fermentation time is 40 days；Fermentation
After the completion, it is separately added into bacillus amyloliquefaciens bacterium solution in the ratio for accessing 10L in every square of mixed fertilizer and marsh is red false single
Born of the same parents' bacterium bacterium solution, adjustment pH value are 7, moisture 60%, and after stirring evenly, booth carries out second at the thin layer of 20cm and ferments, control
Temperature processed is 28 DEG C, after fermentation 8 days, is stirred for uniformly air-drying to moisture content≤15% to get to the special continuous cropping resisting biological of strawberry
Organic fertilizer.
Other methods are the same as 1 the method for embodiment.
Embodiment 3：A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry, after animal dung and edible fungus culturing
Bacteria residue mixing, with bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2, Rhodopseudomonas palustris
（Rhodop seudanonas palustris）With aspergillus niger (AspergillusNige) the bacterium after fermentation edible fungus culturing
The special biologic organic fertilizer of resisting repeated stubbles of the mixture of slag and animal dung, as strawberry, specific preparation method are as follows：
（1）Matrix mixes：Bacteria residue after edible fungus culturing is pressed 1 with cow dung:1 weight ratio is uniformly mixed, and adjusts C/N ratios
It is 30, pH value is adjusted to neutrality, moisture regulation to 65%；
（2）The preparation of microbial inoculum：
A. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 bacterium solutions preparation：The solution of 4 DEG C of preservations
Bacillus amyloliquefaciens JN2 is inoculated on sodium carboxymethylcellulose fluid nutrient medium 37 DEG C, and 150rpm shaking table cultures are stayed overnight, and are prepared into
Seed liquor；
By seed liquor by volume 5% inoculum concentration be inoculated in the sodium carboxymethylcellulose Liquid Culture to sterilize in fermentation tank
In base, tank internal pressure keeps 0.04MPa, 37 DEG C, dissolved oxygen 1.6mg/L, rotating speed 150r/min of temperature, fermented and cultured two days to obtain
JN2 zymotic fluids, it is 4 × 10 to detect living bacteria count in zymotic fluid8cfu/mL；
B. Rhodopseudomonas palustris（Rhodop seudanonas palustris）The preparation of bacterium solution：GH culture mediums are filled
Rhodopseudomonas palustris strain aseptic condition is inoculated in GH culture mediums by whole branch test tube, 30 DEG C of temperature, intensity of illumination 5000Lx
Anaerobic culturel is stood, until bacterium solution becomes peony up to seed liquor；
Rhodopseudomonas palustris is expanded and is cultivated, culture medium and condition of culture are identical as the method for seed liquor is prepared；Work as bacterium
When liquid color becomes laking, obtain Rhodopseudomonas palustris zymotic fluid, detect living bacteria count in zymotic fluid be 4 ×
108cfu/mL；
C. aspergillus niger (AspergillusNige) the preparation of microbial inoculum：Aspergillus niger strain is inoculated in conventional PDA solids training
It supports on base tablet, 28 DEG C of temperature are inverted dark culturing and choose mycelium inoculation in potato glucose liquid after growing fresh mycelia
In culture medium, 28 DEG C, 150rpm shaking table cultures obtain aspergillus niger seed liquor after growing a large amount of hypha bodies in culture medium；
Wheat bran watering is mixed thoroughly, is made the bran mass of moisture content 65%, 110-120 DEG C of high pressure sterilization postcooling,
The ratio that 150mL bacterium solutions are accessed in every kilogram of bran mass accesses aspergillus niger seed liquor, stirs evenly, 28 DEG C of stationary cultures are extremely
A large amount of spores stop culture when generating, and culture is placed in after 30 DEG C of drying in oven up to aspergillus niger bacterium powder；
（3）Microbe inoculation ferments：It is added into mixed fertilizer in the ratio for accessing 10kg in every square metre of mixed fertilizer
Aspergillus niger bacterium powder banks up fermentation after mixing thoroughly, continue to ferment after turning when temperature is less than 50 DEG C, and fermentation time is 45 days；Fermentation
After the completion, it is separately added into bacillus amyloliquefaciens bacterium solution in the ratio for accessing 10L in every square of mixed fertilizer and marsh is red false single
Born of the same parents' bacterium bacterium solution, adjustment pH value are 7, moisture 65%, and after stirring evenly, booth carries out second at the thin layer of 25cm and ferments, control
Temperature processed is 30 DEG C, after fermenting 10 days, is stirred for uniformly air-drying to moisture content≤15% and be given birth to get to the special anti-continuous cropping of strawberry
Organic fertilizer.
Other methods are the same as 1 the method for embodiment.
Experimental example 1：Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 separation identification：
The primary dcreening operation of 1.JN2：The decomposed cow dung sample 10g of acquisition, is put into the triangular flask for filling 90ml sterile waters, shakes
10min is swung, is mixed well, as 10-1Dilution；Take 10-1Dilution 1ml is put into the sterilizing test tubes equipped with 9ml sterile waters,
Concussion, mixes well, as 10-2Dilution；It so repeats, by soil dilution to 10-8Concentration.Take 10-6、10-7、10-8Concentration
Dilution 0.2ml be coated on sodium carboxymethylcellulose solid plate, 37 DEG C, dark condition be inverted culture.Carboxymethyl cellulose
Plain sodium culture medium prescription is：Sodium carboxymethylcellulose 15g/L, yeast powder 1g/L, potassium dihydrogen phosphate 1g/L, ammonium nitrate 1g/
L, magnesium sulfate 0.5g/L.
2. secondary screening：After single bacterium colony is grown, picking single bacterium colony is inoculated in new sodium carboxymethylcellulose solid plate, and 37
DEG C, constant temperature incubation 72h, congo red staining secondary screening filters out the larger bacterium colony of transparent circle, and number is transferred on new tablet, more
Secondary scribing line culture purified is followed by sodium carboxymethylcellulose solid slope, and 4 DEG C preserve for subsequent experimental.
Secondary screening obtains the more plants of larger bacterial strains of transparent circle, selects wherein one plant and cultivates 2 days, as shown in Figure 1, colony diameter
（d）For 2mm, hydrolytic circle（D）For 14mm, D/d values are up to 7, number JN2.Through artificial enrichment culture, isolate and purify
Cultural characteristics of the JN2 on sodium carboxymethylcellulose solid plate is as shown in Fig. 2, be：Colony edge is irregular, radial,
2 ~ 3cm of diameter, light yellow, opaque, surface wettability.JN2 cellular morphologies are observed using simple dyeing and Gram's staining, see figure
3, find the bacterium in rod-shaped.
Its morphological feature is：Colony edge is irregular on sodium carboxymethylcellulose solid plate, radial, diameter
2 ~ 3cm, light yellow, opaque, surface wettability.Gram's staining is positive, rod-shaped, and single, pairs of or bunchiness occurs, and has bud
Spore, sporangium are not expanded, gemma ellipse, middle life.Physiology and biochemistry the results are shown in Table 1.
The physiological and biochemical index of 1 bacterial strain JN2 of table
| Index | As a result | Index | As a result |
| Catalase is tested | + | Hydrolyze starch experiment | + |
| Catalase is tested | + | Methyl red is tested | + |
| D-Glucose is tested | + | Gelatin liquefaction is tested | + |
| L-arabinose is tested | + | Produce hydrogen sulfide experiment | + |
| Mannitol is tested | + | Citric acid is tested |
3.JN2 the Molecular Identification of bacterial strain：The JN2 obtained to screening carries out Molecular Identification as follows：Picking JN2 single bacteriums
It falls and is connected in LB liquid medium, 37 DEG C of shaking table cultures are stayed overnight.200 μ l bacterium solutions, 12000 rpm are taken to centrifuge 5min, abandon supernatant, in
40 μ l sterilizing ultra-pure waters are added in precipitation, after mixing well, are placed in 100 DEG C of water-bath 5min cracking, lysate is carried out as template
PCR reacts.PCR primer is universal primer 27F and 1492R, by raw work bioengineering（Shanghai）Limited liability company synthesizes.Primer
Sequence is：27F：AGA GTT TGA TCC TGG CTC AG and 1492R：TAC GGC TAC CTT GTT ACG ACT T.
PCR system is：10 × PCR Buffer 5 μ L, dNTPs 1.5 μ L, each 0.5 μ L of upstream and downstream primer, 2 μ L, rTaq enzyme of template, 1 μ
L adds water polishing to 50 μ L.PCR programs are 94 DEG C of 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 30 cycles；72℃
5min.PCR product is sent to the sequencing of Beijing Hua Da biotechnology Co., Ltd after the detection of 1.2% agarose gel electrophoresis.Sequence
Row are compared through blast.Through sequencing be accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens)。16SrDNA
As shown in SEQ ID No.1.
According to the result of study to the morphology of bacterial strain, cultural characteristic, Physiology and biochemistry and Molecular Identification, bacterial strain JN2 identifications
For bacillus amyloliquefaciens (Bacillus amyloliquefaciens).Bacillus amyloliquefaciens JN2 is in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center (BeiChen West Road, Chaoyang District, BeiJing City I institutes 3, the micro- life of the Chinese Academy of Sciences
Object research institute) preservation registration number be CGMCC No:10677.
Experimental example 2：Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 is to the main soil-borne disease of strawberry
Harmful preventive and therapeutic effect measures：
1. preventions of the bacillus amyloliquefaciens JN2 to grey mould fruit rot of strawberry：Gray mold is equal in strawberry growth period, florescence, fruiting period
It is likely to occur, pathogen is Botrytis cinerea（Botrytis cinereaPers.）, mycelia, sclerotium and conidium can
It is one of the soil-borne disease of strawberry most serious with overwintering on soil or invalid body or more summer.JN2 fermentations are measured using blade method
Preventive effect of the stoste to grey mould fruit rot of strawberry.Healthy growth, Strawberry Leaves of the same size are cut from petiole, with tap water by leaf
Piece surface washing is clean, then carries out surface sterilization with 70% ethyl alcohol, after being rinsed well with aqua sterilisa, is impregnated in JN2 zymotic fluids
3min dries, and blade face is positioned over upward in the culture dish for being covered with aseptic filter paper moisturizing, and petiole is wrapped up with the sterilizing cotton balls soaked.
Botrytis cinerea mycelia takes toothpick to scratch vaccination ways and is inoculated on blade.Zymotic fluid is replaced with sterile water as a contrast.Every group each
30 blades.Experiment carries out disease survey after 15 days.The state of an illness is remembered with ocular estimate according to the browning situation of every leaf
It carries.
Strawberry leaf diseases are by 5 grades of divisions：0 grade：It is disease-free；1 grade：Onset area accounts for 1/4 or less leaf surface product；2 grades：Morbidity
Area accounts for leaf surface product 1/4-1/2；3 grades：Onset area accounts for leaf surface product 1/2-1/3；4 grades：Onset area accounts for leaf surface product 3/4
More than.
Disease index（%）=。
Experimental result is shown in Table 2, Fig. 4, as a result shows：Control group is only inoculated with botrytis cinerea, and almost all is caught an illness, and tea occurs in blade
Brown scab, scab are gradually expanded, and after 15 days, some whole leafs become dark brown, and surface covers one layer of taupe bacterium
Silk, disease index is up to 87.5%.Experimental group blade first inoculates HM after JN2 is handled, and after 15 days, tea occurs in only a small amount of blade
Brown scab, disease index are only 2.5%.Illustrate that JN2 has higher preventive effect to grey mould fruit rot of strawberry.
Preventive effect of 2 JN2 of the table processing to gray mold
| For trying blade amt（Piece） | Disease index（%） | |
| 0-HM | 30 | 87.5 |
| JN-HM | 30 | 2.5 |
2. bacillus amyloliquefaciens JN2 is to the antagonism of Fusarium and quasi- Pestalotia pathogen：
Fusarium fungus（Fusariumspp.）Worldwide widely distributed, it can endanger various plants, destroy
The fibrovascular system of plant causes plant wilt death and organ to be rotted, and is to prevent most difficult one of soil-borne disease in production.
It can cause droop on strawberry, strawberry root vascular bundle browning, leaf curl is made to wilt until complete stool is dead.Quasi- disk crinosity
Spore category（Pestalotiopsis）It is famous phytopathogen kind, can causes leaf spot on strawberry, main harm blade,
Originally bronzing scab is generated, rear center becomes light brown, generates wheel line, and apparent crineous necrosis band occurs in edge, seriously
When blade it is withered.
Antagonisms of the bacillus amyloliquefaciens JN2 to sickle-like bacteria and quasi- pestalotia bacteria is verified using opposite culture.Sickle
Knife bacterium and quasi- pestalotia bacteria are that this laboratory preserves disease strain, by the fresh mycelium inoculation of disease strain in potato grape
JN2, is inoculated in around disease bacterium, in 28 DEG C of items by sugared solid medium tablets center by the direction on three vertex of equilateral triangle
It is dark under part to be inverted culture.To be only inoculated with disease bacterium, it is not inoculated with the tablet of JN2 as a contrast.Observation disease bacterium growth after 7 days
Situation., it is apparent that compared with the control, sickle-like bacteria and the growth of quasi- pestalotia bacteria for being inoculated with JN2 bacterial strains are apparent from Fig. 5
It is suppressed, illustrates that JN2 has strong antagonism to sickle-like bacteria and quasi- pestalotia bacteria.
Experimental example 3：Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 makees the degradation of cellulose
Detection：
With carboxymethylcelluloenzyme enzyme（CMCase）And filter paper enzyme activity（FPase）For the fermentation condition of index optimization JN2 bacterial strains.
Using national standard 20287-2006《Agricultural microbial agent》In reducing sugar method measure filter paper enzyme activity and carboxymethylcelluloenzyme enzyme
Vigor.With 1mL enzyme solutions, 1min generates 1 μ g glucose and is defined as 1 enzyme activity unit（U）.Standard curve is shown in Fig. 6, recurrence side
Journey is y=0.0002x-0.0223, R2Value is 0.9904, and linear relationship is good.
The variation of 1.JN2 bacterial strains cellulase activity under different fiber-like substrate for induction：With sodium carboxymethylcellulose, filter
Paper slip, corn stalk powder, rice straw powder, sawdust are inducing substrate, prepare different fermentation mediums respectively.Specific basis culture
Based formulas is：Substrate 15g/L, yeast powder 1g/L, potassium dihydrogen phosphate 1g/L, ammonium nitrate 1g/L, magnesium sulfate 0.5g/L take culture
JN2 seed fermentation liquid for 24 hours is inoculated in different fermentation mediums, 37 DEG C, and 160 r/min shaking table cultures centrifuge after 5 days,
It takes supernatant as crude enzyme liquid, measures enzyme activity.
The carboxymethylcelluloenzyme enzyme of different substrates is lived and filter paper enzyme activity value is shown in Fig. 7, it is seen that in sodium carboxymethylcellulose, filter paper
Under the conditions of 5 kinds of item, corn stalk powder, rice straw powder, sawdust inducing substrates, generated carboxymethylcelluloenzyme enzyme work and filter paper enzyme activity
It is variant, wherein the inducibility of maize straw is maximum, and CMC enzyme activity can reach 1344U, and filter paper enzyme activity can reach 835U.Therefore
Select maize straw for best inducing substrate.
The variation of 2.JN2 bacterial strains cellulase activity under condition of different pH：According under different fiber-like substrate for induction
The detection of cellulase activity as a result, using maize straw as inducing substrate, be 3,4,5,6,7,9 to prepare respectively not by pH value
Same fermentation medium.Specific culture medium prescription, condition of culture are examined with cellulase activity under different fiber-like substrate for induction
The culture medium and condition of culture of survey.Culture measures filter paper enzyme activity after 5 days.It the results are shown in Table 3.As it can be seen that when pH value is 7, JN2 is generated
Filter paper enzyme activity highest, 7 be most suitable culture pH conditions.
Filter paper enzyme activity under 3 condition of different pH of table
| pH | 3 | 4 | 5 | 6 | 7 | 9 |
| Filter paper enzyme activity | 793 | 780 | 805 | 739 | 830 | 726 |
3.JN2 bacterial strains different fermentations mode, under the different fermentations time cellulase activity variation：It is trained provided with standing
It supports and two kinds of fermentation methods of shaking table culture, other condition of culture changes with cellulase activity under different fiber-like substrate for induction
Condition of culture takes crude enzyme liquid to measure filter paper enzyme activity and carboxymethylcelluloenzyme enzyme activity when cultivating 5,7,10,12,14 days respectively.
As a result such as table 4, it is seen then that when stationary culture, carboxymethylcelluloenzyme enzyme is lived and filter paper enzyme activity peaked at the 7th day；Shaking table is trained
When supporting, carboxymethylcelluloenzyme enzyme is lived and filter paper enzyme activity peaked at the 10th day.Under two kinds of training methods, the peak of two kinds of enzyme activity
Value difference is not different notable.Therefore, two kinds of training methods can select.
The influence of 4 training method of table and time to cellulase activity
The variation of 4.JN2 bacterial strains cellulase activity at a temperature of different fermentations：Maize straw culture medium is prepared, by difference
The method of cellulase activity variation detection is inoculated with JN2 bacterial strains under fiber-like substrate for induction, in 20 DEG C, 25 DEG C, 37 DEG C, 50 DEG C four
It is left to ferment under a different cultivation temperature, 7 days whens take crude enzyme liquid to measure filter paper enzyme activity and carboxymethylcelluloenzyme enzyme activity.Knot
Fruit sees Fig. 8.It can be seen that：When temperature is 20 DEG C, carboxymethylcelluloenzyme enzyme work highest reaches 1503U；And temperature be 25 DEG C when, Filter paperlyase
Highest living, reaches 910U.Therefore, in fermentation, 20 ~ 25 DEG C are preferably selected.
Experimental example 4：The special biologic organic fertilizer of resisting repeated stubbles of strawberry prepared by the present invention tests the greenhouse of strawberry
2013, be examination material with strawberry cultivars beauty, in the Shanxi province Taiyuan city Yangqu County villages Pu Zi, Pu Feng agriculture and forestry science and technologies have
It is divided to two cells to be tested in one greenhouse of limit company.Continuous 4 years plantation strawberries of the greenhouse, continuous cropping disease incidence are higher.
One cell is processing, uses the special biologic organic fertilizer of resisting repeated stubbles of strawberry prepared by the present invention；Another cell is control, is made
With commercially available composite fertilizer, other way to manage all sames.Consider that pathogenic microorganism can be likely to result in different small with air borne
Section interferes with each other, so setting high barrier partitions two minizones, there are protection zones at greenhouse both ends.Planting patterns is small height
Ridge cultivation is planted, small high ridge top width 50cm, lower width 70cm, the high 30cm of furrow, furrow ditch bottom width 25cm or so, per two row of ridge cultivation, strain spacing 20 ~
25cm.Include 10 ridges per cell.In September plant beauty's detoxic seedling on the 20th, seedling size, growing way are consistent.Respectively at seedling stage, full blossom
Phase, fruiting period investigation strawberry incidence simultaneously calculate disease index and relative control effect；Strawberry increment is investigated in strawberry Later growth
（Using dry matter weight as measurement index）；Yield of strawberry, quality are investigated in fruiting period（2 ridge culture are randomly selected in every processing section
For the fixed area investigated, single plant yield is recorded to end from harvest is started, finally with the average single plant yield of every processing
For computing unit）.
Experimental result：Table 5 is the control effect to continuous cropping greenhouse strawberry soil-borne disease using biological organic fertilizer of the invention.
As can be seen that in the droop, gray mold, the main soil-borne disease of three kinds of leaf spot that we investigate, the incidence of gray mold is most
Height can reach 34.18% in fruiting period highest incidence, using biological organic fertilizer of the invention to the crop field preventive effect of gray mold
Highest can reach 100% preventive effect in seedling stage, can reach 90.23% in fruiting period, effectively control the generation of gray mold.Droop
Generation with leaf spot can also be well controlled, and the anti-efficiency of fruiting period respectively reaches 67.05% and 56.40%.
5 biological organic fertilizer of table is to greenhouse strawberry soil-borne disease control effect
Table 6 is the influence for using the biological organic fertilizer of the present invention and being grown to greenhouse strawberry seedling using Common compound fertilizer, can
To find out, " biological organic fertilizer " handles the Strawberry Seedlings upgrowth situation of cell and the Strawberry Seedlings of Common compound fertilizer processing cell in blade
Significant difference is not shown in terms of number, blade area, cauline leaf dry matter and root system dry matter.But " biology is shown in table 7
Organic fertilizer " handles that cell fruit quality is best, and it is big to show as fruit, and sugar content is high, and titratable acid is low, raciness, in good taste.Cause
This, it is believed that, biological organic fertilizer of the invention is in terms of preventing continuous cropping strawberry cropping disease and improves strawberry fruit quality side
Face performance is preferable.
The influence that 6 biological organic fertilizer of table grows greenhouse strawberry
| Processing mode | The number of blade | Blade area（cm2） | Blade dry matter（g） | Stem dry matter (g) | Root system dry matter (g) |
| Bio-fertilizer | 15a | 709a | 25.2a | 16.4a | 26.5a |
| Composite fertilizer | 13a | 710a | 19.8b | 15.5a | 26.7a |
Note：A, b in same row are indicated in 0.05 horizontal upper significant difference.
Influence of 7 biological organic fertilizer of table to greenhouse strawberry yield and quality
| Processing mode | Average single plant yield（g） | Mean fruit weight（g） | Soluble solid（%） | Total reducing sugar（%） | Titratable acid（%） | Sugar-acid ratio |
| Bio-fertilizer | 240a | 12.8a | 13.7a | 8.3a | 0.8a | 10.4a |
| Composite fertilizer | 223a | 11.0b | 13.2a | 6.4b | 1.1b | 5.8b |
Note：A, b in same row are indicated in 0.05 horizontal upper significant difference.
Sequence table
<110>Agricultural Biotechnology Research Center of Shanxi Province, Research Inst. for fruit Tree, Agricultural Academy of Shanxi Prov., Shanxi
Shanxi Academy of Agricultural Sciences's agricultural resource and the institute for economic research
<120>A kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry
<160> 1
<170> PaUentIn Version 3.5
<210> 1
<211> 1406
<212> DNA
<213>Artificial sequence
<223>Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 16SrDNA sequences
<400> 1
<400> 1
1 AGGTTACCTC ACCGACTTCG GGTGTTACAA ACTCTCGTGG TGTGACGGGC GGTGTGTACA
61 AGGCCCGGGA ACGTATTCAC CGCGGCATGC TGATCCGCGA TTACTAGCGA TTCCAGCTTC
121 ACGCAGTCGA GTTGCAGACT GCGATCCGAA CTGAGAACAG ATTTGTGGGA TTGGCTTAAC
181 CTCGCGGTTT CGCTGCCCTT TGTTCTGTCC ATTGTAGCAC GTGTGTAGCC CAGGTCATAA
241 GGGGCATGAT GATTTGACGT CATCCCCACC TTCCTCCGGT TTGTCACCGG CAGTCACCTT
301 AGAGTGCCCA ACTGAATGCT GGCAACTAAG ATCAAGGGTT GCGCTCGTTG CGGGACTTAA
361 CCCAACATCT CACGACACGA GCTGACGACA ACCATGCACC ACCTGTCACT CTGCCCCCGA
421 AGGGGACGTC CTATCTCTAG GATTGTCAGA GGATGTCAAG ACCTGGTAAG GTTCTTCGCG
481 TTGCTTCGAA TTAAACCACA TGCTCCACCG CTTGTGCGGG CCCCCGTCAA TTCCTTTGAG
541 TTTCAGTCTT GCGACCGTAC TCCCCAGGCG GAGTGCTTAA TGCGTTAGCT GCAGCACTAA
601 GGGGCGGAAA CCCCCTAACA CTTAGCACTC ATCGTTTACG GCGTGGACTA CCAGGGTATC
661 TAATCCTGTT CGCTCCCCAC GCTTTCGCTC CTCAGCGTCA GTTACAGACC AGAGAGTCGC
721 CTTCGCCACT GGTGTTCCTC CACATCTCTA CGCATTTCAC CGCTACACGT GGAATTCCAC
781 TCTCCTCTTC TGCACTCAAG TTCCCCAGTT TCCAATGACC CTCCCCGGTT GAGCCGGGGG
841 CTTTCACATC AGACTTAAGA AACCGCCTGC GAGCCCTTTA CGCCCAATAA TTCCGGACAA
901 CGCTTGCCAC CTACGTATTA CCGCGGCTGC TGGCACGTAG TTAGCCGTGG CTTTCTGGTT
961 AGGTACCGTC AAGGTGCCGC CCTATTTGAA CGGCACTTGT TCTTCCCTAA CAACAGAGCT
1021 TTACGATCCG AAAACCTTCA TCACTCACGC GGCGTTGCTC CGTCAGACTT TCGTCCATTG
1081 CGGAAGATTC CCTACTGCTG CCTCCCGTAG GAGTCTGGGC CGTGTCTCAG TCCCAGTGTG
1141 GCCGATCACC CTCTCAGGTC GGCTACGCAT CGTCGCCTTG GTGAGCCGTT ACCTCACCAA
1201 CTAGCTAATG CGCCGCGGGT CCATCTGTAA GTGGTAGCCG AAGCCACCTT TTATGTCTGA
1261 ACCATGCGGT TCAGACAACC ATCCGGTATT AGCCCCGGTT TCCCGGAGTT ATCCCAGTCT
1321 TACAGGCAGG TTACCCACGT GTTACTCACC CGTCCGCCGC TAACATCAGG GAGCAAGCTC
1381 CCATCTGTCC GCTCGACTTG CATGTA
Claims (3)
1. a kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry, it is characterised in that：By animal dung and edible fungus culturing
Afterwards bacteria residue mixing, with bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2, Rhodopseudomonas palustris
（Rhodop seudanonas palustris）With aspergillus niger (AspergillusNige) the bacterium after fermentation edible fungus culturing
The special biologic organic fertilizer of resisting repeated stubbles of the mixture of slag and animal dung, as strawberry, specific preparation method are as follows：
（1）Fertilizer mixes：By the bacteria residue after edible fungus culturing with cow dung by weight 1:1 is uniformly mixed, and adjustment C/N ratios are 20
~ 30, pH value is adjusted to neutrality, moisture regulation to 55-65%；
（2）The preparation of microbial inoculum：
A. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 bacterium solutions preparation：The solution starch of 4 DEG C of preservations
Bacillus JN2 is inoculated on sodium carboxymethylcellulose fluid nutrient medium 37 DEG C, and 100 ~ 150rpm shaking table cultures are stayed overnight, and are prepared into
Seed liquor；
By seed liquor by volume 1% ~ 5% inoculum concentration be inoculated in the sodium carboxymethylcellulose fluid nutrient medium to sterilize in fermentation tank
In, tank internal pressure keeps 0.02-0.04MPa, 37 DEG C, 1.8 ± 0.2mg/L of dissolved oxygen, rotating speed 150r/min of temperature, fermented and cultured two
It, obtains JN2 zymotic fluids, and it is 4 × 10 to detect living bacteria count in zymotic fluid8cfu/mL；
B. Rhodopseudomonas palustris（Rhodop seudanonas palustris）The preparation of bacterium solution：GH culture mediums fill whole branch
Rhodopseudomonas palustris strain aseptic condition is inoculated in GH culture mediums by test tube, 30 DEG C of temperature, and intensity of illumination 5000Lx is stood
Anaerobic culturel, until bacterium solution becomes peony up to seed liquor；
Rhodopseudomonas palustris is expanded and is cultivated, culture medium and condition of culture are identical as the method for seed liquor is prepared；When bacterium solution face
When discoloration laking, Rhodopseudomonas palustris zymotic fluid is obtained, it is 4 × 10 to detect living bacteria count in zymotic fluid8cfu/mL；
C. aspergillus niger (AspergillusNige) the preparation of microbial inoculum：Aspergillus niger strain is inoculated in common PDA solid mediums
On tablet, 28 DEG C of temperature are inverted dark culturing and choose mycelium inoculation in potato glucose Liquid Culture after growing fresh mycelia
In base, 28 DEG C, 100 ~ 150rpm shaking table cultures obtain aspergillus niger seed liquor after growing a large amount of hypha bodies in culture medium；
Wheat bran watering is mixed thoroughly, is made the bran mass of moisture content 55-65%, 110-120 DEG C of high pressure sterilization postcooling,
The ratio that 150mL bacterium solutions are accessed in every kilogram of bran mass accesses aspergillus niger seed liquor, stirs evenly, 28 DEG C of stationary cultures are extremely
A large amount of spores stop culture when generating, and culture is placed in after 30 DEG C of drying in oven up to aspergillus niger bacterium powder；
（3）Microbe inoculation ferments：Black song is added into mixed fertilizer in the ratio for accessing 10kg in every square metre of mixed fertilizer
Mould powder banks up fermentation after mixing thoroughly, continue to ferment after turning when temperature is less than 50 DEG C, and fermentation time is 30 ~ 45 days；It has fermented
Cheng Hou is separately added into the red false unit cell of bacillus amyloliquefaciens bacterium solution and marsh in the ratio for accessing 10L in every square of mixed fertilizer
Bacterium bacterium solution, adjustment pH value is 6 ~ 7, moisture 55-65%, and after stirring evenly, booth carries out second at the thin layer of 15-25cm
Fermentation after fermentation 7 ~ 10 days, is stirred for uniformly air-drying to moisture content≤15% to get to strawberry controlled at 25 ~ 30 DEG C
Special biologic organic fertilizer of resisting repeated stubbles；
The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JN2 is in Chinese microorganism strain preservation management
Committee's common micro-organisms center preservation, deposit number are：CGMCC No:10677, the deposit date is：It protects on April 1st, 2015
Hide unit address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode are
100101。
2. a kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry according to claim 1, it is characterised in that：Institute
Stating sodium carboxymethylcellulose culture medium prescription is：Sodium carboxymethylcellulose 15g/L, yeast powder 1g/L, potassium dihydrogen phosphate 1g/L, nitre
Sour ammonium 1g/L, magnesium sulfate 0.5g/L；The GH culture medium prescriptions are：Potassium dihydrogen phosphate 1g/L, sodium acetate 1g/L, magnesium sulfate
0.5g/L, ammonium sulfate 1g/L, sodium chloride 1g/L, calcium chloride 0.1g/L, sodium succinate 1g/L, yeast powder 0.5g/L, peptone
0.5g/L, pH value 6.2.
3. a kind of preparation method of the special biologic organic fertilizer of resisting repeated stubbles of strawberry according to claim 1, it is characterised in that：Institute
Total living bacteria count content >=4 × 10 of the special biologic organic fertilizer of resisting repeated stubbles of strawberry of preparation8CFU/mL。
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