CN108102957A - One plant has phosphorus decomposing and bacillus subtilis and its application of high yield polyglutamic acid - Google Patents
One plant has phosphorus decomposing and bacillus subtilis and its application of high yield polyglutamic acid Download PDFInfo
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Abstract
A bacillus subtilis according to the present invention has stronger phosphate solubilization, has relatively production γ polyglutamic acid abilities, and have preferable control effect to plurality of plant diseases by force, and promote the effect of plant growth.The present invention phosphorus decomposing disease prevention growth-promoting effect bacillus subtilis JCD H 16, deposit number be CGMCC NO.13663, preservation date：On 2 16th, 2017, with phosphorus decomposing, disease prevention growth-promoting, high yield γ polyglutamic acids, increasing yield and improving quality, antimicrobial spectrum extensively wait multi-efficiencies, bacterial strain and its microbial inoculum of the present invention can effectively prevent powdery mildew, bacterial wilt, gray mold, rice sheath blight disease, pimento anthracnose, damping-off etc., broad-spectrum antiseptic and stable and durable effect have development set phosphorus decomposing, production γ polyglutamic acids and the bacterial manure and microbial pesticide preparation of controlling disease one.The nitrogenous fertilizer and phosphate fertilizer of half or so can be reduced using the bacillus subtilis bacterial manure of the present invention, also has apparent effect of increasing production.
Description
Technical field
The invention belongs to microbial manures and microbial pesticide technical field, are specifically related to a kind of with phosphorus decomposing diseases prevention rush
Raw effect and the bacillus subtilis JCD-H-16 of high yield gamma-polyglutamic acid.
Background technology
The average content of phosphorus is about 0.122% in the earth's crust, and in soil forming process, phosphorus has a degree of migration, with
The progress of soil-forming process, soil phosphorus content also reduces.China's major part soil phosphorus content between 0.043%-0.046%, because
And in the soil, phosphorus content is to compare shortage.Since phosphorus is one of 16 kinds of nutrients necessary to growth and development of plants, make
Object mainly absorbs phosphorus element with chemical forms such as phosphate radicals, and the phosphorus in soil mainly exists with the minerals state of stationary state, about
70% or so of content of tatal phosphorus is accounted for, so the content height of water-soluble inorganic phosphate radical ion is in Plant P Nutrition in soil
It plays an important role in supply.Soils in south china overwhelming majority slant acidity, general content of tatal phosphorus is less than 0.04%, wherein more than 80%
Phosphorus is indissoluble state, and the soil texture exists mostly in the form of host mineral, a small number of to exist with phosphoric acid alferric mineral state, some
Phosphorus is organophosphor, is existed mostly in the form of organic compound form such as phosphatide, under normal circumstances, it is difficult to directly be absorbed by plants
It utilizes, only in long-term efflorescence or under appropriate conditions, these phosphate are made by the activation of edaphon
With water-soluble state is progressively converted into, utilization can be just absorbed by crops.In the existing 1.24 hundred million hm2 arable lands in China, there is 0.81 hundred million
Hm2 lacks phosphorus, accounts for more than the 60% of total area under cultivation, and present cropping pattern using inorganic phosphorus fertilizer mostly using directly meeting
Crop is to the demand of phosphorus element, since the useful effect form of the phosphate fertilizer of application is phosphate radical, in the soil easily with basic soil
In calcium salt and acid soil in iron, aluminium plasma react, fixed by colloidal substance in soil, so as to change into nothing
State is imitated, is finally fixed in the form of insoluble phosphate or ADSORPTION STATE in soil, thus plant is difficult to directly absorb, so
The utilization rate of phosphate fertilizer is very low, generally below 20%, in order to improve the utilization rate of phosphate fertilizer, reduce the input of phosphate fertilizer, many agriculturals and
Fertilizer researcher employs the utilization rate that many methods improve phosphate fertilizer, but ineffective, wherein utilizing phosphate solubilizing microorganism pair
The decomposition of soil phophorus is to improve one of important channel that soil phophorus validation utilizes.It is mainly concentrated for the bacillus of phosphorus decomposing
In bacillus megaterium, and for other bacillus, also only Wang Huaping discloses one in patent 201410095857.6
Bacillus amyloliquefaciens ZH1 can be grown using insoluble phosphate for phosphorus source, is improved the utilization rate of fertilizer, is promoted plant
Root system development and the absorption to fertilizer increase soil available phosphorus content and the patentee in the patent No. 2015109510983
In disclose one plant have phosphorus decomposing disease prevention growth-promoting effect bacillus amyloliquefaciens.
In recent years, in agricultural production, the bacterial wilt of crop, rice sheath blight disease, powdery mildew of strawberry, the diseases such as cucumber fusarium axysporum
Morbidity is serious, and for these diseases, current pesticide is in safety, effectively, in terms of residual, all there are it is certain the problem of.With people
Raising to environmental protection and food safety requirements, microbial pesticide it is in widespread attention.But micro- life of the prior art
Object pesticide, antibacterial spectral limit is smaller, and fungistatic effect is undesirable.Some domestic units are also to the bacillus subtilis with Biocontrol Effect
Bacterium is studied, and the controlling object being related to has leaf diseases, soil-borne disease and fruit disease of field crop etc..Different strains
Antimicrobial spectrum, bacteriostasis or even antifungal mechanism etc. also have larger difference.The rhizosphere soil from crop, root table, plant and
The more plants of withered grass buds to the fungi of Different Crop and bacteriosis with antagonism his bacillus strains is separated on blade,
And the diseases prevention efficiency of bacterial strain is improved using artificial mutation, these bacterial strains have been carried out with the research of fermentation condition, has been isolated
Some antibacterial materials simultaneously study its characteristic, while have carried out the greenhouse and field experiment of biocontrol agent.The country has opened
The bacillus subtilis commercial preparation sent out successfully and put into production has hundred anti-, wheat Fengning, line Qu Ning, is obtained according to day, root-rot disappears.Cloud
Microbial pesticide " hundred is anti-" (1,000,000,000/g bacillus subtilis wettable powders that southern agriculture university and China Agricultural University develop jointly
Agent) Ministry of Agriculture's registration is obtained, it is promoted the use of in multiple provinces, about 4 667hm2 of popularizing area mainly prevents rice banded sclerotial blight
Disease, notoginseng root rot, tobacco black shank.Hundred anti-principle active components are bacillus subtilis B908.In the application of crop field, hundred is anti-
To rice sheath blight disease preventive effect more than 70%.Its antimicrobial mechanism is nutrient competition, site is captured.The wheat of Agricultural University Of Nanjing's research and development
Fengning is the living body biological fungicide made of bacillus subtilis strain B3, to wheat sharp eyespot field efficacy up to 50% ~
80%, diseases prevention mechanism, which is mainly manifested in generate, inhibits wheat sharp eyespot germ mycelial growth, Sclerotia forming and sclerotial germination
Antibacterial material.The line Qu Ning of Jiangsu Su Ke agrochemical Co., Ltd is 10,000,000,000 living spores/ml bacillus subtilises aqua and 2.5%
The compound preparation of jinggangmeisu, it is main to prevent rice sheath blight disease and false smut.Root-rot disappears to be stepped on for Kunming Wo Lin bio-engineering corporations
The wettable powder compounded by bacillus subtilis and Pseudomonas fluorescens of note registration, Radix Notoginseng root-rot is prevented by root irrigation
Disease.Wuhan Nature completes the anti-ash of bacillus subtilis BS-208 wettable powders registered and gone into operation
Mould, 2 kinds of diseases of white powder are bacterial plant protective agent, are the competitive inhibitors of various plants pathogen.It passes through competitiveness
Growth and breeding occupies the mode of living space to prevent the growth of phytopathogen, can quickly form one layer of protection in plant surface
Film makes crops cause harm from pathogen.Bacillus subtilis can also secrete antibacterial substance, inhibit germ spore-germination and mycelia
Growth.The research by biological control rice disease for many years such as Jiangsu Province Agriculture Science Institute Chen Zhi friendship obtains prevention rice banded sclerotial blight
The effect of disease is widely popularized up to 50.0% ~ 81.0% bacillus subtilis B-916 bacterial strains on Jiangsu and other places.It is black
It is withered that the bacillus subtilis aqua of Longjiang microbe research institute of the academy of sciences of province research and development mainly prevents melon and vegetable at protected field
It withers disease, damping-off and beans root rot, is promoted and applied on Heilungkiang and other places.
Polyglutamic acid（PGA）Mainly it is polymerized by D-Glu and/or Pidolidone by amido bond.Due to polymerization side
Formula is different, and polyglutamic acid is mainly by two kinds of configurations：α-polyglutamic acid（It is polymerize by α-amido bond, α-PGA）With γ-poly- paddy
Propylhomoserin（It is polymerize by γ-amido bond, γ-PGA）.The gamma-polyglutamic acid of biosynthesis is usually by 500~5000 paddy ammonia
Acid monomers form, and relative molecular weight is 10~10000KD.Contain largely free hydrophily carboxyl on γ-PGA molecular backbones,
It can chelate, be crosslinked and the reactions such as derivatization, therefore, there are good water-soluble, biological degradability and hydrolysis properties.
Cell dehydration can be prevented in terms of physiological function, protect cells from the degradation etc. of protease.γ-PGA are used as fertilizer synergist,
With use, increase crop yield, improvement crop quality and the enhancing crop disease-resistant energy that crop nutrition content is promoted to absorb, reduces fertilizer
Power and other effects.Foliage-spray, root, which fill, applies γ-PGA aquas or applies γ-PGA Synergistic composite fertilizers, can increase satsuma orange
Single fruit weight improves content of fruit soluble solids and vitamin content, improves the exterior quality of greenhouse plant citrus, makes the fruit of open country
Real neat and consistent, fruit shape are justified just, and uniform coloring has the effect naturally waxed, and appearance is best, such as improve crop seed hair
Bud rate, enhancing drought resisting, cold-resistant and disease resistance, improve utilization rate of fertilizer, increase crop yield, improving quality, improvement soil and
Water-holding fresh-keeping etc..
γ-PGA are mostly obtained by microbe fermentation method.Presently found γ-PGA productions bacterial strain is mainly bacillus
Belong to, including bacillus subtilis（Bacillus. subtillis）, Bacillus anthracis（B. anthracis）, lichens bud
Spore bacillus（B.licheniformis ）, bacillus pumilus（B. brevis）, heat-resistant bacillus
（B.licheniformis）And bacillus amyloliquefaciens（B. anmloliquefaciens）.Also some researches show that other strains
As the fermentation of Archimycetes, bacterium and eucaryote also can obtain γ-PGA.Whether glutamic acid is added according to culture medium, by γ-PGA
Producing strains are divided into non-glutamic acid and rely on bacterium and glutamic acid dependence two class of bacterium；Chen Liang is disclosed in the patent No. 2015101652435
The bacillus subtilis strain of a variety of antibacterial lipopeptids of one plant of coproduction and gamma-polyglutamic acid is deposited in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, preserving number is CGMCC NO.9377, but the bacterial strain does not have the effect of phosphorus decomposing, arrives
So far, not being disclosed also both has phosphorus decomposing, also has prevention plurality of plant diseases and the withered grass of high yield gamma-polyglutamic acid
Bacillus.
The content of the invention
The object of the present invention is to provide a kind of withered grass gemma with the effect of phosphorus decomposing disease prevention growth-promoting and high yield gamma-polyglutamic acid
Bacillus and its application can also have the microbial pesticide for preventing a variety of soil-borne diseases and leaf class disease as phosphate solubilizing bacterial manure.And
There is preferable control effect to plurality of plant diseases.
The bacillus subtilis JCD-H-16 of the phosphorus decomposing disease prevention growth-promoting effect of the present invention, the strain is in Chaoyang District, Beijing City
No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservations of the institute of North Star West Road 1, deposit number are
CGMCC NO.13663 preservation dates are on 2 16th, 2017；With phosphorus decomposing, disease prevention growth-promoting, increasing yield and improving quality, antimicrobial spectrum is wide, high
Multi-efficiencies, the bacterial strains and its microbial inoculum of the present invention such as production gamma-polyglutamic acid can effectively prevent powdery mildew, bacterial wilt, gray mold, rice
Banded sclerotial blight, pimento anthracnose, damping-off, pimento anthracnose etc., broad-spectrum antiseptic and stable and durable effect have exploitation collected explanations or commentaries
Phosphorus, weight-reducing and the bacterial manure and microbial pesticide preparation of controlling disease one.
It is a further object to provide phosphorus decomposing disease prevention growth-promoting bacillus subtilis JCD-H-12 application comprising with
Lower step：First, culture presevation pipe is taken out, tablet is drawn with LB solid mediums and recovers, when 37 DEG C of cultures 48 are small.Under tablet
Picking single bacterium colony is inoculated in 50 milliliters of LB culture mediums, when 37 DEG C of shake cultures 24 are small in the incubator.Seed connects using 5%
Kind amount is seeded in the big triangular flasks of 5L that liquid amount is 2L LB culture mediums, when 37 DEG C of shake culture 20-28 are small, detects its bacterium solution
Concentration, viable bacteria content are more than 2,000,000,000/milliliter, you can are seeded to 600L production spore fermented and cultureds as bacillus subtilis liquid spawn
Base, inoculum concentration 10%, it is preceding 8 it is small when, open stirring 120r/min, throughput liquid/gas be 1：0.8, then, stirring 180r/min is opened,
Throughput liquid/gas is 1：When 1.5,37 DEG C of culture 28-42 are small, spore content is treated not less than 2,000,000,000/milliliter, gamma-polyglutamic acid contains
Amount is not less than 20 g/L, you can terminates fermentation, sterile filling obtains liquid end product；Or powdery finished product is obtained by spray drying；
2nd, as bacterial manure, with the ratio of 0.1%-0.2% by the bacillus subtilis microbial agent finished product of step 1 preparation before administration
Example is added in the chemical fertilizer such as N P and K and applies together；
3rd, for soil-borne disease such as tobacco bacterial wilt, damping-off etc., bacillus subtilis microbial agent prepared by step 1 dilutes
It is prevented using the method for pouring root after 500-1000 times, in transplanting and early stage medication；
4th, for leaf diseases such as rice sheath blight disease, powdery mildew of strawberry etc., microbial inoculum prepared by step 1 dilutes 300-500 times
It is prevented using the method for spraying, is started medication at the initial stage of a disease, used once every 7 days, it is 3-5 times common；
Wherein described LB culture mediums：Peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium plus
Enter agar 2%；
Wherein, the production spore fermentation medium：30 g/L of bean cake powder, sodium glutamate 30 g/L, glucose 25g/L, corn pulp 3
G/L, fish meal 2g/L, 0.3 g/L of magnesium sulfate, 0.2 g/L of manganese sulfate, three water dipotassium hydrogen phosphates, 0.4 g/L, 0.4 g/L of calcium chloride,
pH7.2.The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
Wherein, the soil-borne disease is bacterial wilt, Rhizoctonia solani Kuhn, black and white wheel branch, loose type mulberry fruit sclerotium, root rot, white
Thin,tough silk disease etc.；
Wherein, the leaf diseases be rice sheath blight disease, the black star of powdery mildew of strawberry, F.graminearum schw, cucumber, bakanae disease of rice
Bacterium, orchid anthrax, water Xanthomonas oryzae pv.oryzae, wax gourd anthrax, anthocaulus rod method, cucumber foxiness, Alternarialongipes, Chinese alpine rush anthrax,
Muskmelon grey mold, cotton anthrax, Chinese cabbage anthrax, aloe anthrax, coral anthrax etc..
Specific embodiment
With reference to specific embodiment, the present invention is further explained.
Embodiment 1：The separation of bacillus with phosphate solubilization
167 bacillus are separated to from Hunan, Changping, Hebei, Liaoning and other places, wherein 101 plants have Decomposing phosphate activity, from
In
Screen 18 plants high of bacterial strain of phosphorus decomposing intensity.By measuring aqueous solution phosphorus in 18 bacillus shake flask fermentation liquid, determine
Capacity of decomposition of each bacterial strain to Phos, organophosphor, such as table 1, from the results, it was seen that solution Phos is not with solving organophosphor
It is positively related, it is comprehensive understand organophosphor and Phos as a result, we determined that wherein five plants of JCD-H-12, JCD-H-16, JCD-
Q-3, JCD-L-4, JCD-L-16 have the bacterial strain of stronger phosphate solubilization.Organophosphor and inorganic phosphorus bacteria are planted using following two
Culture medium detected when culture 48 is small the performance of strains tested phosphorus decomposing in water solubility.
Solve Phos active culture base（g/L）：(NH4) 2SO40.5, MgSO47 H2O 0.5, NaCl 0.2, Ca3
(PO4) 210.0, CaCO30.5, glucose 2.0, MnSO40.02, FeSO40.02, distilled water 1000 mL, pH7.2.
Solve organic phosphorus active culture medium（g/L）：(NH4) 2SO40.5, MgSO47 H2O 0.5, NaCl 0.2,
CaCO30.5, glucose 2.0, MnSO40.02, FeSO40.02, lecithin 0.2,1000 mL of distilled water
The measure of water-soluble phosphorus content in strains tested culture solution is carried out using molybdenum antimony resistance colorimetric method, determines the bacillus phosphorus decomposing
Ability
Bacterial strain dissolving P capacity
1 each bacterium dissolving P capacity in culture solution of table
Implement the screening of the antibacterial tests of 2 bacillus with stronger phosphate solubilization
By JCD-H-12, JCD-H-16, JCD-Q-3, JCD-L-4, JCD-L-16 in pythium spp（Phythium spp.）、
Rice blast（Pyriculavia oryzae cav.）, capsicum early epidemic germ（Alternaria solani
pepper）, tomato bacterial wilt（Psevdomonas solanacearum）, Rhizoctonia solani Kuhn（Rhizoctonia solani）、
Powdery Mildew（Erisyphe spp.）、
Tobacco black shank（Phytophthora parasitica var.nicotianae）, Botrytis cinerea bacterium（Botrytis
cinera Pers.ex Fr.）Wait pathogenic microorganisms tablet on draw " cross ", when 28 DEG C of constant temperature incubations 72 are small after, see whether have
There is inhibition zone, the result is shown in the following table 2.
The antibacterial tests result of the bacillus of table the last 2 phosphate solubilization
The identification of 3 bacillus subtilis JCD-H-16 of embodiment and wide spectrum antibiosis attribute testing
By physiological and biochemical test, gram-positive bacteria is rod-shaped, endogenous spore, atrichia；The shape on beef peptone culture medium
Into milky bacterium colony, surface is smooth, and edge is irregular.Glucose fermentation sample is positive, and Starch Hydrolysis experiment is positive, gelatin liquefaction
Experiment is positive, V.P reacting positives, catalase reacting positive, and clark and Lubsreaction is positive, and nitrate reduction test is positive, citric acid
Salt is using positive, aerobic growth, and optimal pH 6-8.5, growth optimum temperature is 28-38 DEG C, is accredited as by 16s rDNA withered
Careless bacillus.
Ring activation bacterium solution cross is taken to line on PDA plate JCD-H-16, cause of disease is inoculated at away from inoculation line 3cm
Bacterium, 28 DEG C of culture 7d, measures antibacterial bandwidth, result of the test such as the following table 3.
3 JCD-H-16 of table is to the inhibition of various pathogens
When embodiment 4 is due to doing medium optimization, it is found that its culture viscosity is bigger, check in document, possible its has stronger production
The ability of gamma-polyglutamic acid on following gamma-polyglutamic acid fermentation medium, is tested in 500L fermentation tanks, highest γ-poly-
The content of glutamic acid can reach 57.8 g/L, but its viscosity is very big, and bacillus subtilis does not also produce gemma, therefore, look for
Go out a kind of not only high yield gamma-polyglutamic acid but can the culture medium of high yield gemma be more satisfactory；Wherein, gamma-polyglutamic acid ferments
Culture medium：Seven water of 70 g/L of sodium glutamate, 30 g/L of glucose, 10 g/L of sodium citrate, 1 g/L of sodium nitrate, 4 g/L of ammonium chloride
0.5 g/L of magnesium sulfate, 0.5 g/L of white vitriol, three water dipotassium hydrogen phosphates, 0.2 g/L, 0.4 g/L of calcium chloride, sulfuric acid monohydrate
0.02 g/L of manganese.
The acquisition of the microbial inoculum of 5 bacillus subtilis JCD-H-16 of embodiment
Culture presevation pipe is taken out, tablet is drawn with LB solid mediums and recovers, when 37 DEG C of cultures 48 are small.The picking list under tablet
In a 50 milliliters of LB culture mediums of colony inoculation, when 37 DEG C of shake cultures 24 are small in the incubator.Seed uses 5% inoculum concentration, connects
Kind to liquid amount for 2L LB culture mediums the big triangular flasks of 5L in, when 37 DEG C of shake culture 20-28 are small, detect its bacterial concentration,
Viable bacteria content is more than 2,000,000,000/milliliter, you can is seeded to 600L production spore fermentation mediums as bacillus subtilis liquid spawn, connects
Kind of amount for 10%, it is preceding 8 it is small when, open stirring 120r/min, throughput liquid/gas is 1：0.8, then, open stirring 180r/min, ventilation
It is 1 to measure liquid/gas：When 1.5,37 DEG C of culture 28-42 are small, spore content is treated not less than 2,000,000,000/milliliter, gamma-polyglutamic acid content is not
Less than 20 g/L, you can terminate fermentation, sterile filling obtains liquid end product；Or powdery finished product is obtained by spray drying.Wherein
The LB culture mediums：Peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2 add in agar in solid medium
2%.Wherein described production spore fermentation medium：30 g/L of bean cake powder, sodium glutamate 30 g/L, glucose 25g/L, 3 g/ of corn pulp
L, fish meal 2g/L, 0.3 g/L of magnesium sulfate, 0.2 g/L of manganese sulfate, three water dipotassium hydrogen phosphates, 0.4 g/L, 0.4 g/L of calcium chloride,
pH7.2.The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Embodiment 6 is using bacillus subtilis microbial inoculum JCD-H-16 in field to the preventive and therapeutic effect of plant disease
Using the microbial inoculum fermented respectively to the crop of the diseases such as tobacco bacterial wilt, rice sheath blight disease, powdery mildew of strawberry, carry out
Preliminary field control effectiveness test.For root disease（Such as tobacco bacterial wilt）, pouring root is used after microbial inoculum is diluted 500-1000 times
Method prevented（Transplanting when and early stage medication）, total dosage is 1 kg/acre.It observes and counts the hair of crop
Sick rate or disease refer to.The specific effect that pouring root generates is shown in Table, and wherein blank refers to that crop does not take any control measure；Control group is
Common medicine is prevented；Test group prevents for bacillus subtilis microbial inoculum JCD-H-16
For leaf diseases（Such as rice sheath blight disease, powdery mildew of strawberry etc.）, microbial inoculum is diluted into the 300-500 times of method using spraying
It is prevented, is started medication at the initial stage of a disease, used once every 7 days, it is 3-5 times common.Observe and count crop incidence or
Disease refers to.The effect reached of spraying is shown in Table, and wherein blank refers to that crop does not take any control measure, and control group is that common medicament tries
It tests, test group prevents for bacillus subtilis microbial agent JCD-H-16.
4 JCD-H-16 of table is in field to the control effect of all kinds of diseases
Application effect of 7 bacillus subtilis microbial agent of embodiment on eggplant
1. test site：Guizhou Province Fenggang County He Ba townshiies Bai La holes
2. experimental field：3 mu, dosage is base fertilizer per acre：Urea 2kg, Diammonium phosphate (DAP) 5kg（Weight-reducing half）, potassium sulfate 6kg；Fruit
Expanding stage top dressing：Urea 4kg, potassium sulfate 5kg control groups：2 mu, base fertilizer：Urea 4kg, Diammonium phosphate (DAP) 10kg, potassium sulfate 6kg；Fruit
Real expanding stage top dressing：Urea 8kg, potassium sulfate 5kg；Test method：When eggplant is transplanted, with 100 times of bacillus subtilis microbial agent
Dilution carries out viscous root, in eggplant growth period, foliage-spray 1：500 times of microbial inoculums, 20 days 1 time, even spray 3 times；After foliar spray is applied
10 days or so with 1：1000 times of bacterial manure again pouring root once, control group fresh water spraying.After control group finds bacterial wilt, streptomysin is used
Conventional control is carried out with 50% fenaminosulf wettable powder；
3. result of the test：For test group than control group volume increase 30.03%, mu has a net increase of income up to 654 yuan, it is most important that applies withered
After careless gemma bacillus agent, the bacterial wilt of plant considerably reduces, and control group morbidity is than more serious, just appropriate using fungicide
Control the propagation of disease.Test group is in entire application process without using pesticide, greatly reducing planting cost.
Illustrating the bacillus subtilis microbial agent of the present invention has the effect of apparent weight-reducing disease-preventing and yield-increasing.
Application effect of 5 bacillus subtilis microbial agent of table on eggplant
| Bacterial wilt incidence | Disease index | Preventive effect（%） | Yield（kg） | Rate of growth (%) | |
| Test group | 1.9 | 1.4 | 88.89 | 2832 | 30.03 |
| Control group | 16.4 | 12.7 | 2178 |
Claims (4)
1. one plant has phosphorus decomposing disease prevention growth-promoting effect and high yield gamma-polyglutamic acid bacillus subtilis Bacillus
Subtilis JCD-H-16, the strain is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese microorganism strain preservation pipe
The common micro-organisms center preservation of the reason committee, deposit number are CGMCC NO.13663, and preservation date is on 2 16th, 2017.
2. the application of the one plant according to claim 1 bacillus subtilis with phosphorus decomposing disease prevention growth-promoting effect, feature
It is that the application of the bacillus subtilis of phosphorus decomposing disease prevention growth-promoting and high yield gamma-polyglutamic acid comprises the steps of：First, bacterium is taken out
Kind preservation pipe draws tablet with LB solid mediums and recovers, when 37 DEG C of cultures 48 are small；Picking single bacterium colony is inoculated under tablet
In 50 milliliters of LB culture mediums, when 37 DEG C of shake cultures 24 are small in the incubator；Seed uses 5% inoculum concentration, is seeded to liquid amount
In the big triangular flasks of 5L of 2L LB culture mediums, when 37 DEG C of shake culture 20-28 are small, to detect its bacterial concentration, viable bacteria content is more than
2000000000/milliliter, you can be seeded to 600L production spore fermentation mediums as bacillus subtilis liquid spawn, inoculum concentration 10% is preceding
8 it is small when, open stirring 120r/min, throughput liquid/gas be 1：0.8, then, stirring 180r/min is opened, throughput liquid/gas is 1：
When 1.5,37 DEG C of culture 28-42 are small, spore content is treated not less than 2,000,000,000/milliliter, gamma-polyglutamic acid content is not less than 20 g/L,
It can terminate to ferment, sterile filling obtains liquid end product；Or powdery finished product is obtained by spray drying；
2nd, as bacterial manure, with the ratio of 0.1%-0.2% by the bacillus subtilis microbial agent finished product of step 1 preparation before administration
Example is added in the chemical fertilizer such as N P and K and applies together；
3rd, for soil-borne disease such as tobacco bacterial wilt, damping-off etc., bacillus subtilis microbial agent prepared by step 1 dilutes
It is prevented using the method for pouring root after 500-1000 times, in transplanting and early stage medication；
4th, for leaf diseases such as rice sheath blight disease, powdery mildew of strawberry etc., microbial inoculum prepared by step 1 dilutes 300-500 times
It is prevented using the method for spraying, is started medication at the initial stage of a disease, used once every 7 days, it is 3-5 times common；
Wherein described LB culture mediums：Peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium plus
Enter agar 2%；
Wherein described production spore fermentation medium：30 g/L of bean cake powder, sodium glutamate 30 g/L, glucose 25g/L, corn pulp 3
G/L, fish meal 2g/L, 0.3 g/L of magnesium sulfate, 0.2 g/L of manganese sulfate, three water dipotassium hydrogen phosphates, 0.4 g/L, 0.4 g/L of calcium chloride,
pH7.2；The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
3. the application of the one plant according to claim 2 bacillus subtilis with phosphorus decomposing disease prevention growth-promoting effect, feature
It is, the soil-borne disease is bacterial wilt, Rhizoctonia solani Kuhn, black and white wheel branch, loose type mulberry fruit sclerotium, root rot, southern blight
Deng.
4. the application of the one plant according to claim 2 bacillus subtilis with phosphorus decomposing disease prevention growth-promoting effect, feature
Be, the leaf diseases be rice sheath blight disease, the black star of powdery mildew of strawberry, F.graminearum schw, cucumber, fusarium moniliforme, orchid
Flower anthrax, water Xanthomonas oryzae pv.oryzae, wax gourd anthrax, anthocaulus rod method, cucumber foxiness, Alternarialongipes, Chinese alpine rush anthrax, muskmelon ash
Mould, cotton anthrax, Chinese cabbage anthrax, aloe anthrax, coral anthrax etc..
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