CN108823140A - A kind of withered bioconversion microbial bacterial agent of paulownia and preparation method thereof for preventing and treating yellow twig - Google Patents
A kind of withered bioconversion microbial bacterial agent of paulownia and preparation method thereof for preventing and treating yellow twig Download PDFInfo
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Abstract
The present invention provides a kind of withered bioconversion microbial bacterial agent of paulownia and preparation method thereof for preventing and treating yellow twig, includes the following steps：Take mellow soil drying and screening, it is added containing the withered domestication culture medium solution with honey of paulownia, successive shaking flask and stationary culture, remove liquid portion, domestication culture medium solution is added, repeats to cultivate, sediment fraction is added in LB culture medium and is shaken up, bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are obtained through dilution spread separation, culture, is mixed to get microorganism fungus kind for four；It is grafted to containing in the withered withered fermentation liquid of paulownia with honey of paulownia, limits aerobe fermentation, filtering obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.The withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig prepared by the present invention is made using the withered Promoting bacteria fermentation withered raw material of paulownia of paulownia, is had and is killed wood louse, kills pathogen, induction plant broad spectrum antidisease and the multiple-effect for promoting plant inorganic nutrients and leaf photosynthesis.
Description
Technical field
The invention belongs to yellow twig control agent technical fields, and in particular to a kind of withered bioconversion of paulownia for preventing and treating yellow twig is micro-
Bacteria agent and preparation method thereof.
Background technique
Citrus is the important cultivated plant in the whole world, and cultivated area and yield position are at the forefront in the world, but with global citrus
The development of industry, but citrus pest and disease damage industry has propagated to the main production district of the citruses such as Asia, Africa, America, especially citrus Huanglong
Disease, the disease infect fast, and disease incidence is high, have resulted in more than one hundred million plants of mandarin trees and catch an illness or dead.Huanglong's encephalapthy agent is bast bar
Bacterium, gramnegative bacterium, thallus is most rounded or oval, and minority is in irregular shape, size be 20-600nm ×
170-1600nm.The outer limit of thallus is film quality structure, thickness about 17-33nm, average 25nm.The 16S of Huanglong's encephalapthy agent
RDNA sequence belongs to Gracilicutes α subclass, and is divided into Asia kind Las, Africa kind of Laf and America kind Lam, Asia kind Las and beauty
Continent kind Lam belongs to heat resistant type, and Africa kind of a Laf belongs to responsive type, and different types of Huanglong's encephalapthy agent can cause citrus bast
Tissue necrosis and screen casing blocking, cause metabolic disorder, decline even dead.
Yellow twig can also be propagated mainly using diaphorina citri as communication media by grafting, Semen Cuscutae, therefore yellow twig is anti-
To control method mainly include using nontoxic propagation material, roots out cause of disease tree and prevention and control wood louse.Chinese patent CN 104604946B is disclosed
A kind of biologic protein agents and preparation method thereof for prevention and control Citrus Huanglongbing pathogen, said preparation is by streptomyces hygroscopicus ACCC
40473 strains are activated, expand culture medium fermentation tank culture stops fermentation acquisition water suction strepto- when reaching 60-80% to cell concentration
Bacterium, by streptomyces hygroscopicus carry out clasmatosis concentration remove moisture removal, obtain concentrated broth, by concentrated broth, NAC albumen,
Diatomite/kaolin/bentonite carrier and neopelex/lauryl sodium sulfate auxiliary agent be uniformly mixed, xeraphium
It is broken, the biologic protein agents powder for prevention and control Citrus Huanglongbing pathogen is obtained, the auxiliary agent of this method preparation is living by antibiotic and biology
The microbial metabolic products of property substance are also right to Citrus Huanglongbing pathogen by good preventive effect as active principle reasonable compatibility
Diseased plant is by certain recovery effects.A kind of microbial bacterial agent preventing yellow twig disclosed in Chinese patent CN 106350464A and
Preparation method, the microbial bacterial agent include Gu Lake streptomycete fermentation liquid, Ka Wuer streptomycete fermentation liquid, the sugared gemma bar of cold solution
Fermented liquid, Nissl fermentation of bacillus liquid and tuft wood fermentation liquid, the microbial bacterial agent pass through foliage-spray and simultaneously pouring root,
Monthly twice using one, inhibit the reproduction of gramnegative bacterium to multiply with this, kill harmful bacteria of soil, in addition the microorganism
Microbial inoculum can generate the antiinsect antibiotics such as yellow Nysfungin, inhibition or the development of failure line wood louse and other insect vector worm's ovums, subtract
Few adult is green, reduces orchard pest occurrence quantity, cuts off the communication media of yellow twig, have the function that prevention and control Citrus Huanglongbing pathogen.By
The above-mentioned prior art is it is found that fungus treatment has certain control efficiency to yellow twig, and the present invention is by plant extracts and microbial inoculum
With the use of in the prevention and treatment for Citrus Huanglongbing pathogen, obtaining good control efficiency.
Summary of the invention
The technical problem to be solved in the present invention is to provide it is a kind of prevent and treat yellow twig the withered bioconversion microbial bacterial agent of paulownia and
Preparation method, using the withered bacillus subtilis in honey domestication culture soil of paulownia, bacillus amyloliquefaciens, short and small gemma
Bacillus and bacillus licheniformis, then by bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacterium grafts on containing oxygen culture is limited in the withered withered fermentation liquid of paulownia with honey of paulownia, obtains the withered bioconversion microorganism of paulownia of prevention and treatment yellow twig
Microbial inoculum.The withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig prepared by the present invention, which has, to be killed wood louse, kills pathogen, lures
It leads plant broad spectrum antidisease and promotes the multiple-effect of plant inorganic nutrients and leaf photosynthesis.
In order to solve the above technical problems, the technical scheme is that：
A kind of preparation method of the withered bioconversion microbial bacterial agent of paulownia that preventing and treating yellow twig, it is characterised in that：Include the following steps：
（1）After taking mellow soil drying and screening, it is added containing the withered domestication culture medium solution with honey of paulownia, first in 30 DEG C and 200r/
Under conditions of min, for 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion for shaking flask culture, adds containing paulownia is withered and bee
The domestication culture medium solution of honey repeats culture 9 times, takes sediment fraction；
（2）By step（1）The sediment fraction of preparation is added in LB culture medium and shakes up, through the isolated bacillus subtilis of dilution spread
Bacterium, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis, by bacillus subtilis, bacillus amyloliquefaciens, short
Bacillus pumilus and bacillus licheniformis individually use LB culture medium under conditions of 30 DEG C and 200r/min, shaking flask culture
12-24h obtains bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis mixed configuration micro-
Biological inoculum；
（3）Containing inoculation step in the withered withered fermentation liquid of paulownia with honey of paulownia（2）The microorganism fungus kind of preparation limits aerobe fermentation 5-10d, mistake
Filter obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
As a preferred embodiment of the above technical solution, the step（1）In, mellow soil is healthy old tung oil tree and wild health
Soil under surface layer within the scope of Fructus Aurantii tree tree crown at 15-30cm, the partial size of mellow soil are 40-100 mesh.
As a preferred embodiment of the above technical solution, the step（1）In, containing paulownia in the withered domestication culture medium solution with honey of paulownia
Withered content is 50g/L, and the content of honey is 10g/L.
As a preferred embodiment of the above technical solution, the step（1）In, mellow soil with containing paulownia is withered and the domestication culture of honey
The materials ratio of based sols is 1g：100mL.
As a preferred embodiment of the above technical solution, the step（2）In, the materials ratio of sediment fraction and LB culture medium is 1g：
100mL。
As a preferred embodiment of the above technical solution, the step（2）In, bacillus subtilis, solution starch in microorganism fungus kind
The mass ratio of bacillus, bacillus pumilus and bacillus licheniformis is 1:1:1:1.
As a preferred embodiment of the above technical solution, the step（3）In, it is withered containing paulownia in the withered withered fermentation liquid of paulownia with honey of paulownia
Content is 10-100g/L, and the content of honey is 10-50g/L, and it be partial size is 40-100 mesh powder that paulownia is withered.
As a preferred embodiment of the above technical solution, the step（3）In, the dosage of microorganism fungus kind accounts for the 1- of total system quality
5%。
As a preferred embodiment of the above technical solution, the step（3）In, the aperture of filtering is 100 mesh.
As a preferred embodiment of the above technical solution, the user of the withered bioconversion microbial bacterial agent of paulownia of the prevention and treatment yellow twig
Method is：After 100-500 times of dilution agent of the paulownia for preventing and treating yellow twig withered bioconversion microbial bacteria, foliage-spray, trickle irrigation root are carried out
Portion, or the root to plant, skin, leaf, fruit and limb carry out spraying treatment.
It is appropriate highly concentrated in the critical period that the bud phase and leaf stretching period for being easy to be infected are using three-dimensional control prece
Degree, which uses, plays an important roll the disease control in entire growth period, and due to the withered life of paulownia of prevention and treatment yellow twig prepared by the present invention
The green safe property of object microbial microbial inoculum is good, therefore appropriate high concentration uses, and will not damage to strain body；It is bred in germ
And after hiding autumn fruit picking, sprays on a large scale again, can effectively solve coming year cause of disease radix and disease hazard degree.In addition,
Pouring root, Qing Yuan (limb is spraying), spraying mode carry out three-dimensional chemoprevention to the root of tree body, skin, leaf, fruit during spraying
It controls, makes entire tree body that can absorb the effective ingredient of medical fluid, hiding bast and overwintering is effectively killed in conductive process
Germ.
In addition, the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig prepared by the present invention can be with the prevention and treatment side of yellow twig
Method common implementing.
The control method of yellow twig includes：
1. strictly quarantine system：It is incoming without lesion and novel species growing area to prevent sick seedling, sick fringe.
2. cultivating disease-free nursery stock：
1. nursery lot should select in no lesion or the good place of isolation condition, or with the closed nursery of plastic wire canopy.2. establishing mandarin orange
The virus-free breeding of method of tangerine.All breeding strains through selecting, it is necessary to be examined by indicator plant or polymerase chain reaction (PCR)
It surveys.Stem apex nursery stock is obtained by Shoot-tip Grafting detoxification technology, breeds disease-free nursery stock by virus-free guidelines.3. " system " is not built
Immediately, stock seed should pick up from the fruit of no sick tree, seed with 50 ~ 52 DEG C hot-water soak 5 minutes, be immersed in 55 after preheating again ~
In 56 DEG C of hot water, constant temperature was up to 50 minutes.Scion should pick up from identified disease-free elite stand, and with 1 000 times of quadracycline liquid
It impregnates 2 hours, it is rear to rinse grafting well with clear water.4. reinforcing the management system in nursery.
3. preventing and treating diaphorina citri：
1. reinforcing cultivation management, each young sprout takes out hair neatly, unified spray prevention and treatment.First time pesticide is sprayed in the budding period, is prevented
Adult worms producting eggs are controlled at bud gap.Isocarbophos, flolimat or machine oil emulsion etc. can be selected based on organic phosphorus in pesticide.2. plus
Strong winter clear garden spray, eliminates the diaphorina citri of Wintering Period energy difference, is the key that annual prevention and treatment.3. adjusting fruit variety knot
Structure does not plant the rutaceaes such as Calusena lansium, kamuning in citrus producing region, prevents diaphorina citri alternate hose.4. yellow twig is tight
The area of weight carries out unified prevention and treatment.
4. it puts prevention first, integrated control：Adhere to checking Huanglong diseased plant comprehensively after each young sprout turns green, one plant of discovery excavates one
Strain, does not stay stub, rationally utilizes physics and biological synthesis method.
5. lesion rebuilds citrus orchard：
1. full wafer is answered to excavate disease, veteran, environment is cleared up, first plants after 1 year emergency crops row kind of citrus again.2. using transad
It plants.
6. developing Orange Producing in a manner of industrialization management, accomplish development scale unified planning, unified kind in garden is raw
Unified measure in production, technical unified instruction, unified regulation, effectively control disease incidence in management.
Compared with prior art, the invention has the advantages that：
（1）The present invention is used as plant source using paulownia is withered, and it is remainder after the oil expression of tung oil tree that paulownia is withered, contains protein nearly 30%,
Starch-containing and soluble sugar is rich in the necessary a great number of elements of plants such as phosphorus calcium and magnesium potassium 25% or so, can be by plant as fertilizer
It absorbs, in addition, also containing terpene, flavonoids, steroid, Coumarins during paulownia is withered.The Secondary metabolites such as alkaloids,
With certain pest-resistant bactericidal effect, there is certain killing effect to wood louse and bast bacillus.But simple paulownia is withered to Huang
The limited and traditional botanical pesticide of the control efficiency of imperial disease is mostly used organic solvent or decocts to extract, and being unfavorable for energy conservation also has
Biggish environmental risk, influence active material stability and lose active material, increase cost make development and application difficulty compared with
Greatly, function and effect are slow, it is difficult to large-scale promotion.The present invention selects paulownia withered and honey is as domestication culture medium, with the old of health
Soil under surface layer within the scope of tung oil tree and wild healthy Fructus Aurantii tree tree crown at 15-30cm is raw material, and repeated culture optimization obtains
To bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis, by bacillus subtilis, Xie Dian
Afnyloliquefaciens, bacillus pumilus and bacillus licheniformis remix to obtain plant growth-promoting bacteria after being separately cultured, which promotees
The preparation method of raw bacterium can be improved thallus yield, shorten fermentation time and reduce production cost, then by the plant growth-promoting of preparation
Bacterium fermentation paulownia is withered, not only contributes to the extraction of the withered middle physiological activator of paulownia, is also less prone to cause the inactivation of active material, and growth-promoting
For bacterium itself there are also preferable disinsection prophylaxis and growth-promoting functions, being more advantageous to improves plant growth and enhancing insecticidal effect, is conducive to
Environmental protection and withered effective use of paulownia itself.
（2）The withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig prepared by the present invention, which has, to be killed wood louse, kills disease
Opportunistic pathogen, induction plant broad spectrum antidisease, it is highly-safe, it can suitably high concentration use, can't be damaged to strain body, and have
It being absorbed conducive to the root of strain body, skin, leaf, fruit, surface and internal progress three-dimensional prevention and treatment to strain body, control efficiency is significant, and
The multiple-effect for promoting plant inorganic nutrients and leaf photosynthesis is conducive to the yield and quality for improving citrus etc..
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be described in detail, herein illustrative examples and explanation of the invention
For explaining the present invention, but it is not as a limitation of the invention.
Embodiment 1：
（1）The mellow soil under the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health at 15cm is taken, it is dry,
After crossing 40 meshes, according to mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：100mL is added
The withered domestication culture medium solution with 10g/L honey of paulownia containing 50g/L, first under conditions of 30 DEG C and 200r/min, shaking flask culture
For 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion, adds containing paulownia is withered and the domestication culture medium solution of honey,
Culture 9 times is repeated, sediment fraction is taken.
（2）It is 1g according to the materials ratio of sediment fraction and LB culture medium：Sediment fraction is added in LB culture medium 100mL
It shakes up, through the isolated bacillus subtilis of dilution spread, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are individually used LB culture medium by bacterium
Under conditions of 30 DEG C and 200r/min, mass ratio is 1 by shaking flask culture 12h:1:1:1 bacillus subtilis solves starch bud
Spore bacillus, bacillus pumilus and bacillus licheniformis mixed configuration obtain microorganism fungus kind.
（3）Microbe inoculation strain in the withered fermentation liquid of paulownia of the withered powder of the 40 mesh paulownia containing 10g/L and 10g/L honey, wherein micro-
The dosage of biological inoculum accounts for the 1% of total system quality, and it be partial size is 40 mesh powders that paulownia is withered, limits aerobe fermentation 5d, filters through 100 mesh,
Obtain the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
Embodiment 2：
（1）The mellow soil under the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health at 30cm is taken, it is dry,
After sieving with 100 mesh sieve, according to mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：100mL is added
The withered domestication culture medium solution with 10g/L honey of paulownia containing 50g/L, first under conditions of 30 DEG C and 200r/min, shaking flask culture
For 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion, adds containing paulownia is withered and the domestication culture medium solution of honey,
Culture 9 times is repeated, sediment fraction is taken.
（2）It is 1g according to the materials ratio of sediment fraction and LB culture medium：Sediment fraction is added in LB culture medium 100mL
It shakes up, through the isolated bacillus subtilis of dilution spread, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are individually used LB culture medium by bacterium
Under conditions of 30 DEG C and 200r/min, mass ratio is 1 for 24 hours by shaking flask culture:1:1:1 bacillus subtilis solves starch bud
Spore bacillus, bacillus pumilus and bacillus licheniformis mixed configuration obtain microorganism fungus kind.
（3）Microbe inoculation strain in the withered fermentation liquid of paulownia of the withered powder of the 100 mesh paulownia containing 100g/L and 50g/L honey, wherein
The dosage of microorganism fungus kind accounts for the 1-5% of total system quality, and it be partial size is 100 mesh powders that paulownia is withered, aerobe fermentation 10d is limited, through 100
Mesh filtering, obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
Embodiment 3：
（1）The mellow soil under the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health at 20cm is taken, it is dry,
After crossing 50 meshes, according to mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：100mL is added
The withered domestication culture medium solution with 10g/L honey of paulownia containing 50g/L, first under conditions of 30 DEG C and 200r/min, shaking flask culture
For 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion, adds containing paulownia is withered and the domestication culture medium solution of honey,
Culture 9 times is repeated, sediment fraction is taken.
（2）It is 1g according to the materials ratio of sediment fraction and LB culture medium：Sediment fraction is added in LB culture medium 100mL
It shakes up, through the isolated bacillus subtilis of dilution spread, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are individually used LB culture medium by bacterium
Under conditions of 30 DEG C and 200r/min, mass ratio is 1 by shaking flask culture 20h:1:1:1 bacillus subtilis solves starch bud
Spore bacillus, bacillus pumilus and bacillus licheniformis mixed configuration obtain microorganism fungus kind.
（3）Microbe inoculation strain in the withered fermentation liquid of paulownia of the withered powder of the 80 mesh paulownia containing 50g/L and 25g/L honey, wherein micro-
The dosage of biological inoculum accounts for the 2% of total system quality, and it be partial size is 60 mesh powders that paulownia is withered, limits aerobe fermentation 8d, filters through 100 mesh,
Obtain the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
Embodiment 4：
（1）The mellow soil under the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health at 15cm is taken, it is dry,
After sieving with 100 mesh sieve, according to mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：100mL is added
The withered domestication culture medium solution with 10g/L honey of paulownia containing 50g/L, first under conditions of 30 DEG C and 200r/min, shaking flask culture
For 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion, adds containing paulownia is withered and the domestication culture medium solution of honey,
Culture 9 times is repeated, sediment fraction is taken.
（2）It is 1g according to the materials ratio of sediment fraction and LB culture medium：Sediment fraction is added in LB culture medium 100mL
It shakes up, through the isolated bacillus subtilis of dilution spread, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are individually used LB culture medium by bacterium
Under conditions of 30 DEG C and 200r/min, mass ratio is 1 by shaking flask culture 18h:1:1:1 bacillus subtilis solves starch bud
Spore bacillus, bacillus pumilus and bacillus licheniformis mixed configuration obtain microorganism fungus kind.
（3）Microbe inoculation strain in the withered fermentation liquid of paulownia of the withered powder of the 100 mesh paulownia containing 10g/L and 50g/L honey, wherein
The dosage of microorganism fungus kind accounts for the 3.5% of total system quality, and it be partial size is 40 mesh powders that paulownia is withered, aerobe fermentation 6d is limited, through 100 mesh
Filtering obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
Embodiment 5：
（1）The mellow soil under the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health at 25cm is taken, it is dry,
After sieving with 100 mesh sieve, according to mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：100mL is added
The withered domestication culture medium solution with 10g/L honey of paulownia containing 50g/L, first under conditions of 30 DEG C and 200r/min, shaking flask culture
For 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion, adds containing paulownia is withered and the domestication culture medium solution of honey,
Culture 9 times is repeated, sediment fraction is taken.
（2）It is 1g according to the materials ratio of sediment fraction and LB culture medium：Sediment fraction is added in LB culture medium 100mL
It shakes up, through the isolated bacillus subtilis of dilution spread, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are individually used LB culture medium by bacterium
Under conditions of 30 DEG C and 200r/min, mass ratio is 1 by shaking flask culture 16h:1:1:1 bacillus subtilis solves starch bud
Spore bacillus, bacillus pumilus and bacillus licheniformis mixed configuration obtain microorganism fungus kind.
（3）Microbe inoculation strain in the withered fermentation liquid of paulownia of the withered powder of the paulownia of mesh containing 75g/L50 and 35g/L honey, wherein micro-
The dosage of biological inoculum accounts for the 1.5% of total system quality, and it be partial size is 40 mesh powders that paulownia is withered, aerobe fermentation 10d is limited, through 100 mesh mistakes
Filter obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
Embodiment 6：
（1）The mellow soil under the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health at 30cm is taken, it is dry,
After crossing 40 meshes, according to mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：100mL is added
The withered domestication culture medium solution with 10g/L honey of paulownia containing 50g/L, first under conditions of 30 DEG C and 200r/min, shaking flask culture
For 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion, adds containing paulownia is withered and the domestication culture medium solution of honey,
Culture 9 times is repeated, sediment fraction is taken.
（2）It is 1g according to the materials ratio of sediment fraction and LB culture medium：Sediment fraction is added in LB culture medium 100mL
It shakes up, through the isolated bacillus subtilis of dilution spread, bacillus amyloliquefaciens, bacillus pumilus and lichens gemma bar
Bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis are individually used LB culture medium by bacterium
Under conditions of 30 DEG C and 200r/min, mass ratio is 1 for 24 hours by shaking flask culture:1:1:1 bacillus subtilis solves starch bud
Spore bacillus, bacillus pumilus and bacillus licheniformis mixed configuration obtain microorganism fungus kind.
（3）Microbe inoculation strain in the withered fermentation liquid of paulownia of the withered powder of the 40 mesh paulownia containing 100g/L and 10g/L honey, wherein
The dosage of microorganism fungus kind accounts for the 5% of total system quality, and it be partial size is 40 mesh powders that paulownia is withered, aerobe fermentation 10d is limited, through 100 mesh mistakes
Filter obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
After sediment fraction culture prepared by embodiment 1, morphology and life are carried out through the isolated product of dilution spread
Manage biochemical character identification and 16S rRNA identification.
Wherein, morphology and physiological and biochemical property identification method are：According to《The outstanding bacterium handbook of uncle》,《Common bacteria identification
Handbook》With《Microbiology Experiment study course》（The second edition）In method strain to be tested is lined into LB solid medium, at 30 DEG C
2 d of lower culture, utilize the form of electron microscope observation bacterial strain.
Bacillus 16S rRNA identification method：Using 16S rRNA gene sequencing method, illustrate to extract by kit
DNA of bacteria and with universal primer F27（5'-AGAGTTTGATCCTGGCTCAG-3'）And R1492（5'-TACGGCTACCTTGTTA
CG ACTT-3'）Bacterial 16 S rRNA gene is expanded, amplified production transfers to raw work bioengineering（Shanghai）Co., Ltd is surveyed
Sequence, sequencing results carry out homologous comparison analysis by the BLAST in NCBI, choose high homology sequence, utilize MEGA4.0
Software building phylogenetic tree.Adjacent method is used in the building of phylogenetic tree, and is repeated sampling 1000 times and carried out value point of bootstrapping
Analysis determines the position of strain during evolution with the confidence level of assessment system development tree.
Bacterial strain hb15 forms circular colonies on LB culture medium, and edge is irregular, and early growth period surface is smooth, later period surface
It is coarse, there is fold, thallus is rod-shaped, Gram-positive, and size is 0.5 μ m, 1.0 μm of ol, to hold raw flagellum movement, shape
At gemma.
CR amplification obtains the 16SrDNA nucleotide fragments of bacterial strain hb15, size 1438bp.It is homologous to be carried out BLAST
Property sequence alignment result show the sequence and 16S rDNA sequence that the multiple bacterial strains of bacillus have been delivered（Such as Bacillus
Licheniformis strain Xmb047 and Bacillus licheniformis strain ML103A）Similitude reach
Physiology and biochemistry is combined to 99% or more as a result, determining that the bacterial strain is bacillus.
Then it is incubated overnight the bacterial strain of preservation using NB culture medium, collects thallus, extracts examination using raw work bacterial genomes
Agent box extracts strain gene group DNA, with primer 2 7F（5′-AGAGTTTGATCCTGGCTCAG-3′）,1492R（5′-
CGGTTACCTTGTTACGACTT-3′）For upstream and downstream primer, pass through the 16SrRNA gene of each bacterial strain of PCR amplification.PCR reactant
System is：2 μ L of DNA profiling, 10 × PCRBuffer, 5 μ L, 4 μ L of 2.5mM dNTP, each 2 μ L of 10 μM of upstream and downstream primers, Taq
2 μ L of enzyme, 35 μ L of distilled water.PCR reaction condition is：95 DEG C of 5 min, 94 DEG C of 30 s, 55 DEG C of 40 s, 72 DEG C of 90 s,
72 DEG C of 5 min, 32 circulations.Take 5 μ L PCR product electrophoresis detections, after remaining product purification with cloning vector pMD 18T
Carrier is connected, and is transferred in e. coli jm109 and is sent to the raw work in Shanghai and is sequenced.By each bacterial strain 16SrRNA gene sequencing number
BLASTN comparison is carried out according in the website NCBI, retrieves homologous sequence, and use adjacent method with MEGA 4.0.2 software
(Neighbor-Joiningmethod) building of systematic evolution tree is carried out, to determine position of the strain in evolution.And it will be each
The 16SrRNA gene sequence data of bacterial strain is submitted to GenBank database, is determined as bacillus subtilis, solution starch gemma bar
Bacterium, bacillus pumilus and bacillus licheniformis.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (10)
1. a kind of preparation method for the withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig, it is characterised in that：Including following step
Suddenly：
（1）After taking mellow soil drying and screening, it is added containing the withered domestication culture medium solution with honey of paulownia, first in 30 DEG C and 200r/
Under conditions of min, for 24 hours, the then stationary culture 48h at 30 DEG C removes liquid portion for shaking flask culture, adds containing paulownia is withered and bee
The domestication culture medium solution of honey repeats culture 9 times, takes sediment fraction；
（2）By step（1）The sediment fraction of preparation is added in LB culture medium and shakes up, through the isolated bacillus subtilis of dilution spread
Bacterium, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis, by bacillus subtilis, bacillus amyloliquefaciens, short
Bacillus pumilus and bacillus licheniformis individually use LB culture medium under conditions of 30 DEG C and 200r/min, shaking flask culture
12-24h obtains bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis mixed configuration micro-
Biological inoculum；
（3）Containing inoculation step in the withered withered fermentation liquid of paulownia with honey of paulownia（2）The microorganism fungus kind of preparation limits aerobe fermentation 5-10d, mistake
Filter obtains the withered bioconversion microbial bacterial agent of paulownia of prevention and treatment yellow twig.
2. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（1）In, mellow soil is the surface layer within the scope of the old tung oil tree and wild healthy Fructus Aurantii tree tree crown of health
Soil at lower 15-30cm, the partial size of mellow soil are 40-100 mesh.
3. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（1）In, the withered content containing paulownia in the withered domestication culture medium solution with honey of paulownia is 50g/L, honey
Content is 10g/L.
4. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（1）In, mellow soil with containing paulownia is withered and the materials ratio of the domestication culture medium solution of honey is 1g：
100mL。
5. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（2）In, the materials ratio of sediment fraction and LB culture medium is 1g：100mL.
6. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（2）In, bacillus subtilis in microorganism fungus kind, bacillus amyloliquefaciens, bacillus pumilus and
The mass ratio of bacillus licheniformis is 1:1:1:1.
7. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（3）In, the withered content containing paulownia in the withered withered fermentation liquid of paulownia with honey of paulownia is 10-100g/L, honey
Content is 10-50g/L, and it be partial size is 40-100 mesh powder that paulownia is withered.
8. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（3）In, the dosage of microorganism fungus kind accounts for the 1-5% of total system quality.
9. a kind of preparation method of withered bioconversion microbial bacterial agent of paulownia for preventing and treating yellow twig according to claim 1,
It is characterized in that：The step（3）In, the aperture of filtering is 100 mesh.
10. a kind of withered bioconversion microbial bacterial agent of the paulownia of prevention and treatment yellow twig described in claim 1-9, which is characterized in that institute
The application method of the withered bioconversion microbial bacterial agent of paulownia for stating prevention and treatment yellow twig is：The withered bioconversion of paulownia for preventing and treating yellow twig is micro-
Bacteria agent dilute 100-500 times after, progress foliage-spray, trickle irrigation root, or the root to plant, skin, leaf, fruit and limb into
Row spraying treatment.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108220185A (en) * | 2017-12-19 | 2018-06-29 | 华中农业大学 | A kind of application of biological control agent in citrus treelet yellow twig cause of disease is removed |
| CN109601237A (en) * | 2019-02-20 | 2019-04-12 | 易之泰生物科技（龙岩）有限公司 | The control method of Citrus Huanglongbing pathogen |
| CN110476692A (en) * | 2019-09-23 | 2019-11-22 | 成都环美园林生态股份有限公司 | A kind of hair oriental cherry method for planting in the above plateau city of four kms |
| CN110495339A (en) * | 2019-09-23 | 2019-11-26 | 成都环美园林生态股份有限公司 | A kind of hall crabapple flower method for planting on the above plateau of four km of height above sea level |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220185A (en) * | 2017-12-19 | 2018-06-29 | 华中农业大学 | A kind of application of biological control agent in citrus treelet yellow twig cause of disease is removed |
| CN109601237A (en) * | 2019-02-20 | 2019-04-12 | 易之泰生物科技（龙岩）有限公司 | The control method of Citrus Huanglongbing pathogen |
| CN110476692A (en) * | 2019-09-23 | 2019-11-22 | 成都环美园林生态股份有限公司 | A kind of hair oriental cherry method for planting in the above plateau city of four kms |
| CN110495339A (en) * | 2019-09-23 | 2019-11-26 | 成都环美园林生态股份有限公司 | A kind of hall crabapple flower method for planting on the above plateau of four km of height above sea level |
| CN110476692B (en) * | 2019-09-23 | 2022-04-08 | 成都环美园林生态股份有限公司 | Method for planting prunus tomentosa in plateau city with height of more than four kilometers |
| CN110495339B (en) * | 2019-09-23 | 2022-04-08 | 成都环美园林生态股份有限公司 | Malus halliana planting method at altitude of more than four kilometers |
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Effective date of registration: 20191203 Address after: Room 101, 160-10 Qian Rong Road, Binhu District, Wuxi City, Jiangsu Province Applicant after: Liu Li Address before: 214000 No. 6106 house, No. 20, No. 20, Po Dong Road, Tianhe District, Wuxi, Jiangsu. Applicant before: Guangzhou Nong Kang Biology Technology Co., Ltd. |
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Application publication date: 20181116 |