CN105638744B - A kind of preparation method of Brevibacillus brevis wettable powder - Google Patents
A kind of preparation method of Brevibacillus brevis wettable powder Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
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- A01N25/14—Powders or granules wettable
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Abstract
The present invention relates to biological pesticide and microbial manure technical field, specifically disclosed is a kind of wettable powder of Brevibacillus brevis containing gemma germinant and preparation method thereof, and the raw material of the wettable powder is made of following composition by weight：40 70 parts of Brevibacillus brevis zymotic fluid；Filler：20 30 parts of white carbon；Wetting agent：14 parts of lauryl sodium sulfate；Dispersant：T-shaped 0.2 1 parts of lignin；Gemma germinant：0.1 0.5 parts of inosine, 0.1 0.5 parts of fructose；Uv-protector：0.5 2 parts of lecithin, 0.2 1 parts of β dextrin；Drying protectant：14 parts of maltose, 0.2 1 parts of sucrose.Gemma speed of germinating is fast after Brevibacillus brevis wettable powder prepared by the present invention applies crop field, and germination rate is high, can significantly improve its fertilizer efficiency and the control effect to plant disease, reduces the dosage of short gemma, reduces production cost.
Description
Technical field
The present invention relates to biological pesticides and microbial manure technical field, and in particular to one kind contains short gemma germinant
Brevibacillus brevis wettable powder and preparation method thereof.
Background technology
China using substantial amounts of chemical pesticide preparation and chemical fertilizer, easily causes pesticide residue exceeded every year, biological concentration, no
Only pollution of ecological environment has an effect on the health of the mankind.And biological pesticide Small side effects, it is harmless to humans, be it is a kind of have it is wide before
The environment friendly agricultural of scape has become a kind of trend of global pesticide development.
Bacillus is a kind of microorganism of current biological pesticide and most study in bio-feritlizer, it has broad-spectrum antiseptic
Active and extremely strong anti-adversity ability, vegetative cell can be multiplied in fungal pathogen mycelium surface to be colonized, and effectively inhibits germ
Growth, therefore, before control disease occurs, plant strain growth, raising crop yield etc. is promoted to show wide application
Scape.
Brevibacillus brevis is a kind of isolated microorganism in the soil, by the study found that its to it is a variety of it is dynamic, plant
Object pathogenetic bacteria and fungi are such as：Escherichia coli, chicken Pasteurella, Staphylococcus aureus, septicaemia bacterium, cotton wilt
Bacterium, verticillium wilt, cucumber fusarium axysporum, Pestallozzia theae, tobacco ralstonia solanacearum etc. have stronger antagonism.
Chen Li etc., which has studied, is isolated from the Brevibacillus brevs A57 bacterial strain of Cotton rhizosphere soil to cotton rhizoctonia solani, withered
It withers the antagonism of germ and verticillium wilt pathogen, the opposite culture on PDA plate, average inhibition is 33. 5 %, 39.5 respectively
%、29.5 %.By micro- sem observation, the main antagonism for finding antagonistic bacterium is can result in by generating antagonistic substance
The fracture of disease fungus mycelium, distortion, deformity, inhibit conidial sprouting, cause spore lopsided.Using wilt as instruction
Bacterium has studied the bacterium solution fermentation condition during strains expressed optimal antagonism activity, the results showed that, A57 appropriate medias are trained for LB
Base is supported, pH8, bacteriostatic activity is best under aerobic conditions.The thick leach protein of A57 bacterial strains is extracted with ammonium sulfate precipitation method, to for examination
Pathogen still has higher antagonistic activity, its antagonistic activity is maximum during 70% concentration, and A57 thick leach proteins are to withered pathogenic bacteria
Maximal percentage inhibition is 23.7%, at the same the supernatant of crude protein that is obtained after ammonium sulfate precipitation of the bacterium to cause of disease indicator bacteria still
With certain antagonistic activity, illustrate A57 antagonistic bacterium antagonisms not only has high molecular weight protein class substance to also have small point
Sub- antagonistic substance.
Zhu Zhongbin etc. studies tobacco rhizosphere biocontrol bacterial strain Brevibacillus brevis（Brevibacillus brevis）DZQ3 pairs
Tobacco growth-promoting effect and inducible system resistance, carry out pot experiment in greenhouse, and DZQ3 is inoculated in a manner of soaking root and add pouring root.With
The method for measuring each processing cigarette strain physical signs, economical character, biomass including cigarette strain, tobacco leaf chlorophyll content, electricity
Solve relative permeability, superoxide dismutase, peroxidase, polyphenol oxidase, phenylalanine lyase and catalase
Wait system resistants index of correlation and root system development situation, influences of the evaluation inoculation DZQ3 to cigarette strain.The result shows that after DZQ3 processing
Tobacco agronomy character and biomass are better than control, and when 30 d is more notable（p<0.05）；System resistant relative physiologic index is better than
Control, when 60 d, are the most notable（p<0.05）.Biocontrol microorganisms DZQ3 has facilitation to the growth and development of tobacco, can induce and is
System resistance enhances the disease resistance of tobacco.
Flavine virtue waits isolated one plant of Brevibacillus brevis from soil（Brevibacillus brevis）JK-2,
Some researches show that it to withered germ of water-melon, verticillium dahliae, balsam pear anthrax bacteria, Monilinia fructicola, the blue or green withered thunder Er Shi of tomato
Bacterium, fowl enteropathogenic E. Coli K88 bacterial strains all have strong inhibitory action, are one plant of biocontrol microorganisms with broad spectrum antibiotic activity,
Through Toxicity Determination, the zymotic fluid of JK-2 has good control effect to banana blight bacteria.Gold is tended to have to wait in tobacco green grass or young crops
Blight area takes health tobacco plant, and 2 plants are separated to out of its cane to tobacco Ralstonia solanacarum (Ralstonia
Solanacarum) there is the interior raw Brevibacillus brevis bacterial strain 009 and 011 of strong antagonism.The indoor leaching root method difference of temperature
009 and 011 bacterial strain is first inoculated with, is inoculated with pathogen afterwards, preventive effect is respectively 87.25% and 52.30%.With 009 and 011 bacterium solution point
Not and the mixed liquor of tobacco ralstonia solanacearum leaching root, preventive effect are significantly lower than the former.Results of field plot shows 011 bacterial strain
Preventive effect apparently higher than 009 bacterial strain and agricultural streptomycin.Huang Lifeng etc. discloses one kind in patent No. 201410519415.X
Brevibacillus brevis H3 and its application in decomposing agent is prepared.Zhou Lihong etc. is disclosed in the patent No. 201410003514.2
One plant of Brevibacillus brevis bacterial strain, the cultural method of bacterial strain and its application in terms of deodorization.Hu Xueqin etc. is in the patent No.
The preparation method of one plant of Brevibacillus brevis for preventing fungal diseases of plants and biological pesticide is disclosed in 201010607988.X,
It can significantly reduce melon droop.Ye Jianren etc. discloses a kind of bacillus brevis in the patent No. 201210111439.2
And its application in pine tree growth is promoted.Lin Birun discloses a kind of prevention capsicum epidemic disease in the patent No. 201110030249.3
Preparation method and the application of the bacillus brevis and biological agent of disease.Gold is tended to have to wait disclosed in the patent No. 201410013801.1
A kind of preparation method of interior raw Brevibacillus brevis wettable powder, by Brevibacillus brevis zymotic fluid, talcum powder, lignin
Sulfoacid calcium, DBS, CMC-Na, dextrin and white carbon form by a certain percentage.Che Jianmei etc. is in the patent No. 201010296437.6
A kind of new Brevibacillus brevis bacterial strain and its application are disclosed, which carries out longan fresh-keeping microorganism
Preserving fungus agent, the microbial inoculum can reduce the rotting rate of longan, and can keep the color and luster and taste of fruit.
Although the microbial pesticide product prepared based on bacillus is many, all things considered, also existing includes research
Exploitation disconnects with production, formulation difficulty, and product quality stability is poor, production technology falls behind, and homogeneity of product is not high, production enterprise
The problem of industry small scale, application difficulty is big, in addition to a little, bacillus is based on wettable powder at present, this dosage form
Although convenient for storage and transport, there are many restrictive factors in production application.As application wettable powder in it is most
Using gemma as the unitary basis for weighing active ingredient in product, if gemma cannot be sprouted in time, application effect is bad, such as king
During pretty any of several broadleaf plants droop Brazilian using bacillus subtilis TR21 preventions, pre-stage test utilizes TR21 bacterial strains NB fermentation liquid irrigating roots and leaf
Armpit inoculation can effectively prevent droop, but using gemma prepare wettable powder pouring root and axil effect it is undesirable,
It demonstrates the pure gemma after removing zymotic fluid and may be because that sprouting is difficult and declines preventive effect.
Bacillus is widely distributed in nature, and when external condition is suitble to growth, brood cell can be passed through by dormant state
A series of complex biochemical reactions grow into trophosome and recover metabolism, this process is known as sprouting
(germination).Gemma sprouting is influenced by exogenous nutrition substance in nature, and this kind of nutriment is referred to as " sprouting
Agent ", usual nutrition induction gemma sprouting include the single effect of amino acid, carbohydrate and fast cry of certain animals nucleosides, also include their collaboration
Effect (such as：AGFK；A：Mono- asparagus fern phthalein amine of L；G：Glucose；F：Fructose；K：Potassium ion).When nutriment touches gemma
When, a series of variations can occur for gemma inside,（1）, H inside gemma coreTenAnd Zn2+Release, cause gemma core internal pH by
Faintly acid rises to alkalescent；(2), Ca inside gemma core2+-- a large amount of releases of DPA；(3), gemma core is hydrated, internal Ca2+--
DPA is substituted by water, and the heat resistance of gemma declines；(4), the gemma skin glycan layer hydrolysis of cortex, gemma swelling of nucleus；(5)、
Gemma core is further hydrated, and gemma core internal protein mobility enhancing, enzymatic activity is strengthened, and synthesizes ATP；(6), degradation SASP eggs
In vain, to discharge gemma DNA, and then the synthesis of RNA, protein, DNA in gemma growth course are guided.
After the gemma germinant mixing that gemma and promotion are sprouted within the several seconds, germinant is quickly across gemma clothing and cortex
The surface of intine is reached, is interacted with the receptor protein on intine, excitation gemma enters germinating, connects
It and starts a succession of sprouting event, germination process afterwards will not stop when removing germinant.
In Bacillus, Bacillus subtillis, thuringiensis, Bacillus anthracis and Bacillus cereus
It is concerned when closely bound up with the production and living of the mankind.It is a series of that research shows that the genome of Bacillus subtillis includes
With antibiotic and the relevant gene cluster of essential industry enzyme, Bacillus anthracis and Bacillus cereus have people and animals very strong
Pathogenic, since the former is for manufacturing biological weapons, and the latter generates the toxin for causing people's vomiting and diarrhea, therefore the two moment prestige
The health and lives safety of the mankind is coerced, they do not possess infection ability when in gemma state, but when into sprouting shape
Start the ability with infection people and animals after state, and finally generate the toxin of pathogenic even causing death；And the pathogenetic bacteria of insect
Thuringiensis has special toxic action to insect, can cause septicemia after the in vivo germination of host,
And their brood cell can effectively improve the virulence of insecticidal crystal protein, and the sprouting of brood cell may be necessary in synergistic effect
's.Therefore, it is pathogenic to inhibit its to the research of bacillus germination process, safeguard that human health and life security have to pass
Important theory significance and actual application value, therefore these four gemma sprout it mechanism and study and must compare with relevant germinant
It is more, but the gemma germinant in relation to Brevibacillus brevis yet there are no disclosure.Do not have in Brevibacillus brevis wettable powder yet
The report of the middle research for adding in gemma germinant.
Further, since the compatibility of the auxiliary agents such as different bacillus and wetting agent, dispersant, carrier is to all kinds of gemma bars
Bacterium adsorptivity is different, and the formula of existing bacillus wettable powder or preparation process are also not used to prepare to contain
The Brevibacillus brevis wettable powder of gemma germinant；The adsorbance of effective bacterium, survival rate, to harmful bacteria in wettable powder
Control effect and wettable powder dosage stability difference it is larger, the preparation method of entire wettable powder must be examined totally
Consider each carrier adsorption ability, to protective effect in the drying process of Brevibacillus brevis, wetting agent, dispersant is to wettable powder
Suspensibility, the influence of MEBO ribbon gauze, and comprehensive screening is carried out to gemma germinant just to access MEBO ribbon gauze short hangs
Floating rate is high, and the gemma holding time is long, using rear gemma energy fast-germination, the good Brevibacillus brevis wettable powder of preventive effect.
The content of the invention
In view of the above shortcomings of the prior art, a kind of Brevibacillus brevis wettable powder containing gemma germinant is provided
And preparation method thereof, after gemma germinant can make Brevibacillus brevis wettable powder incorporate water, short gemma energy fast-germination,
And the germination rate of short gemma is greatly improved, the Brevibacillus brevis wettable powder spore content prepared is not less than 100
Hundred million CFU/ grams, suspensibility is not less than 85%, and wetting time is not higher than 80S, and finess disintegrating crosses 400 mesh sieves, and moisture is not higher than
5%, preparation method is stablized.
In order to achieve the above object, a kind of following weight of Brevibacillus brevis wettable powder is grouped into：Short gemma bar
40-70 parts of fermented liquid；Filler：20-30 parts of white carbon；Wetting agent：1-4 parts of lauryl sodium sulfate；Dispersant：It is T-shaped wooden
It is 0.2-1 parts plain；Gemma germinant：0.1-0.5 parts of inosine, 0.1-0.5 parts of fructose；Uv-protector：0.5-2 parts of lecithin, β-
0.2-1 parts of dextrin；Drying protectant：1-4 parts of maltose, 0.2-1 parts of sucrose.
A kind of preparation method of Brevibacillus brevis wettable powder, is made of suddenly the following steps：
First, the preparation of Brevibacillus brevis zymotic fluid
Culture presevation pipe is taken out, tablet is drawn with LB solid mediums and recovers, when 30 DEG C of cultures 48 are small.It is chosen under tablet
Single bacterium colony is taken to be seeded in 50 milliliters of LB culture mediums, when 30 DEG C of shake cultures 24 are small in the incubator.Seed connects using 5%
Kind amount, the bacillus liquid culture medium being seeded in the big triangular flasks of 5L, liquid amount 2L, when 30 DEG C of shake culture 16-28 are small,
Its bacterial concentration is detected, bacterium solution content is more than 6,000,000,000/milliliter, you can as Brevibacillus brevis seed liquor.By short gemma bar
Bacterium seed liquor is seeded in the fermentation tank of 1000L, and liquid amount is the fermentation medium of 600L, and throughput is every point when being preceding 16 small
Clock liquid-gas ratio is 1：0.6, throughput is 1 thereafter：1；Open stirring, rotating speed 160r/min；It is 30 DEG C when 24 is small before cultivation temperature,
Thereafter, temperature improved 1 DEG C, when culture 28-36 is small when every 4 is small, detects its spore content more than 8,000,000,000 CFU/ml, you can terminate
Fermentation, as Brevibacillus brevis zymotic fluid；
Wherein, the LB culture mediums：Peptone 10g, yeast extract 5g, sodium chloride 10g, water 1000mL, pH7.2, solid training
It supports and agar 2% is added in base.Wherein, the bacillus liquid culture medium：Glucose 10g/L, 8 g/L of soluble starch, beans
15 g/L of dregs of rice powder, 5 g/L of fish meal, 0.5 g/L of magnesium sulfate, 0.5 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, dipotassium hydrogen phosphate
0.5 g/L, pH7.2.Wherein, the fermentation medium：Glucose 5g/L, 20 g/L of corn flour, 50 g/L of bean cake powder are beautiful
10 g/L of rice milk powder, 0.5 g/L of magnesium sulfate, 0.5 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, 0.5 g/L of dipotassium hydrogen phosphate,
pH7.2.The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
2nd, by 40-70 parts of Brevibacillus brevis zymotic fluid；Filler：20-30 parts of white carbon；Uv-protector：Lecithin
0.5-2 parts, 0.2-1 parts of powder-beta-dextrin；Drying protectant：1-4 parts of maltose, 0.2-1 parts of sucrose, is spray-dried after mixing, obtains
Brevibacillus brevis pulvis；
3rd, by wetting agent：1-4 parts of lauryl sodium sulfate；Dispersant：T-shaped lignin 0.2-1 parts；Gemma germinant：
0.1-0.5 parts of inosine, 0.1-0.5 parts of fructose；It is uniformly mixed so as to obtain the auxiliary agent of Brevibacillus brevis；
4th, step 2 and the obtained Brevibacillus brevis pulvis of step 3 and auxiliary agent mixing and mistake are passed through into air-flow crushing
Machine crushed 400 mesh sieves, detect its spore content not less than 10,000,000,000 cfu/g, suspensibility is not less than 85%, and wetting time is not higher than
80s, water content are not higher than 5%, vacuum packaging, you can obtain Brevibacillus brevis wettable powder.
Wherein, Brevibacillus brevis wettable powder optimum weight part composition is：
60 parts of Brevibacillus brevis zymotic fluid；Filler：25 parts of white carbon；Wetting agent：3 parts of lauryl sodium sulfate；It is scattered
Agent：T-shaped 0.5 part of lignin；Gemma germinant：0.2 part of inosine, 0.4 part of fructose；Uv-protector：1.5 parts of lecithin, β-paste
0.8 part of essence；Drying protectant：3 parts of maltose, 0.6 part of sucrose.
Gemma sprouting is influenced by exogenous nutrition substance in nature, and this kind of nutriment is referred to as " germinant ", is led to
In the case of often, after the gemma germinant mixing of gemma and promotion sprouting within the several seconds, germinant is quickly across gemma clothing and skin
Layer reaches the surface of intine, interacts with the receptor protein on intine, and excitation gemma enters germinating,
Then a succession of sprouting event is started, germination process afterwards will not stop when removing germinant.Wettable powder of the present invention
In contain gemma germinant：Inosine, fructose are from glycosides propylhomoserin, alanine, valine, leucine, isoleucine, serine, Soviet Union
Propylhomoserin, methionine, cystine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, phenylalanine,
Tyrosine, tryptophan, histidine, proline, inosine, 2,6 pyridine dicarboxyl calcium carry out single factor experiment and two-by-two group in fructose
What conjunction property was tested has the germinant that short gemma is substantially induced to sprout.Through overtesting, Brevibacillus brevis of the invention
Wettable powder presses 1 with water：100 ratio carries out wetting and is uniformly mixed, and gemma germinant at this time can be contacted fully with gemma
Afterwards, so as to promote its fast-germination, through overtesting, after 30 minutes, gemma germination rate has reached more than 80%, and does not add gemma
After 4 hours, gemma germination rate has only reached 50% or so to the short gemma of germinant.
The present inventor passes through to carrier（Such as bentonite, talcum powder, kaolin, diatomite, white carbon, ground phosphate rock, lightweight carbon
Sour calcium）, auxiliary agent（Wetting agent and dispersant）（Polyvinyl alcohol, polyethylene glycol, CMC-Na, Tween-60, neopelex,
T-shaped lignin, lauryl sodium sulfate, Tea Saponin, Morwet EFW, Morwet D425, My100, D110, Arab's power glue,
2- naphthalene sulfonic acid formaldehyde polymer sodium salts, octyl phenol polyoxyethylene ether, diisooctyl Disodium sulfosuccinate, methylcellulose, wood
Quality sodium sulfonate）Filler（Diatomite, kaolin, bentonite, white carbon, precipitated calcium carbonate, talcum powder）, ultraviolet protective agent
（Ascorbic acid, dextrin, neopelex is Congo red, carboxymethyl cellulose, lecithin）, drying protectant（Defatted milk
Powder, sucrose, sodium glutamate, maltose, mannose, methylcellulose etc.）It is screened, each auxiliary agent is to Brevibacillus brevis
Gemma survival, suspension, wettability tested, and Brevibacillus brevis is determined with orthogonal test finally by more wheel single factor tests
The formula of wettable powder.By verification, Brevibacillus brevis wettable powder of the invention preserves 1 year at 20-25 DEG C,
Its gemma survival rate also can reach 9,000,000,000 CFU/ grams.
The wettable powder of the present invention has adopted vacuum packaging, reduces contact of the gemma with air, reduces the machine of its sprouting
Rate.
The moisture of the wettable powder of the present invention is less than 5%, even if containing bud in the wettable powder of low moisture content
Spore germinant（Inosine and fructose）, the gemma activity in its storage process is not also influenced significantly, it is not reduced and is storing
Gemma during depositing.
Compared with prior art, the present invention haing the following advantages and effect：
This is domestic for the first time by the gemma germinant addition Brevibacillus brevis wettable agent progress industry of Brevibacillus brevis
Metaplasia is produced, and is greatly improved gemma and is sprouted rate and germination rate, is solved because gemma is sprouted slowly, is prevented caused by germination rate is low
The problem of effect is poor, and effect is unstable is controlled, reduces the dosage of Brevibacillus brevis, reduces production cost；
A kind of its spore content of the detection containing gemma germinant is provided not less than 10,000,000,000 cfu/g, suspensibility is not less than 85%,
Wetting time is not higher than 80s, and moisture is less than 5%, the preparation method of vacuum-packed Brevibacillus brevis wettable powder.
Specific embodiment
Substantive distinguishing features of the present invention can be embodied from the following examples, but these embodiments are only used as explanation, without
It is to limit the invention.
Embodiment 1
A kind of following weight of Brevibacillus brevis wettable powder is grouped into：60 parts of Brevibacillus brevis zymotic fluid；Filler：
25 parts of white carbon；Wetting agent：3 parts of lauryl sodium sulfate；Dispersant：T-shaped 0.5 part of lignin；Gemma germinant：Inosine
0.2 part, 0.4 part of fructose；Uv-protector：1.5 parts of lecithin, 0.8 part of powder-beta-dextrin；Drying protectant：3 parts of maltose, sucrose
0.6 part.
A kind of preparation method of Brevibacillus brevis wettable powder, is made of suddenly the following steps：
First, the preparation of Brevibacillus brevis zymotic fluid
Culture presevation pipe is taken out, tablet is drawn with LB solid mediums and recovers, when 30 DEG C of cultures 48 are small.It is chosen under tablet
Single bacterium colony is taken to be seeded in 50 milliliters of LB culture mediums, when 30 DEG C of shake cultures 24 are small in the incubator.Seed connects using 5%
Kind amount, the bacillus liquid culture medium being seeded in the big triangular flasks of 5L, liquid amount 2L, when 30 DEG C of shake culture 16-28 are small,
Its bacterial concentration is detected, bacterium solution content is more than 6,000,000,000/milliliter, you can as Brevibacillus brevis seed liquor.By short gemma bar
Bacterium seed liquor is seeded in the fermentation tank of 1000L, and liquid amount is the fermentation medium of 600L, and throughput is every point when being preceding 16 small
Clock liquid-gas ratio is 1：0.6, throughput is 1 thereafter：1；Open stirring, rotating speed 160r/min, before cultivation temperature 24 it is small when for 30 DEG C,
Thereafter, temperature improved 1 DEG C, when culture 30 is small when every 4 is small, its spore content is detected as 8,400,000,000 CFU/ml, terminates fermentation, as
Brevibacillus brevis zymotic fluid；
Wherein, the LB culture mediums：Peptone 10g, yeast extract 5g, sodium chloride 10g, water 1000mL, pH7.2, solid training
It supports and agar 2% is added in base.Wherein described bacillus liquid body culture medium：Glucose 10g/L, 8 g/L of soluble starch, dregs of beans
15 g/L of powder, 5 g/L of fish meal, 0.5 g/L of magnesium sulfate, 0.5 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, dipotassium hydrogen phosphate
0.5 g/L, pH7.2；
Wherein, the fermentation medium：Glucose 5g/L, 20 g/L of corn flour, 50 g/L of bean cake powder, corn starch
10 g/L, 0.5 g/L of magnesium sulfate, 0.5 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, dipotassium hydrogen phosphate 0.5 g/L, pH7.2；
The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
2nd, by 60 parts of Brevibacillus brevis zymotic fluid；Filler：25 parts of white carbon；Uv-protector：1.5 parts of lecithin, β-
0.8 part of dextrin；Drying protectant：3 parts of maltose, 0.6 part of sucrose, is spray-dried after mixing, obtains Brevibacillus brevis pulvis
3rd, by wetting agent：3 parts of lauryl sodium sulfate；Dispersant：T-shaped 0.5 part of lignin；Gemma germinant：Inosine
0.2 part, 0.4 part of fructose；It is uniformly mixed so as to obtain the auxiliary agent of Brevibacillus brevis
4th, step 2 and the obtained Brevibacillus brevis pulvis of step 3 and auxiliary agent mixing and mistake are passed through into air-flow crushing
Machine crushed 400 mesh sieves, detect its spore content as 10,500,000,000 cfu/g, suspensibility 88%, wetting time 69s, water content
For 4.5%, vacuum packaging, you can obtain Brevibacillus brevis wettable powder.
Embodiment 2
Using Brevibacillus brevis wettable powder in field to the control effect of watermelon blight
Test site：XiaoShan, HangZhou City, Zhejiang Province area farm, long-term planting greenhouse watermelon, due to throughout the year and, disease phase
To heavier, particularly droop；
Test method：9th area of greenhouse point, per 66 square meter of area, each Zu Zuosange parallel tests area；
Test group：Before transplanting, with 100 times of vacuole roots 2 of Brevibacillus brevis wettable powder containing gemma germinant it is small when；
It is sprayed for the first time, and with Brevibacillus brevis wettable powder with 250 times of liquid of Brevibacillus brevis wettable powder within 30 days after transplanting
1000 times of dilution liquid irrigating roots of agent are once；
Control group one：It is small with 100 times of vacuole roots 2 of Brevibacillus brevis wettable powder of no gemma germinant before transplanting
When；It is sprayed for the first time, and with no bud with 250 times of liquid of Brevibacillus brevis wettable powder of no gemma germinant within 30 days after transplanting
1000 times of dilution liquid irrigating roots of Brevibacillus brevis wettable powder of spore germinant are once；
Control group two：That is blank control group, the method identical with test group operation, is not added with Brevibacillus brevis wettable powder
Agent is compareed with clear water.
Result of the test：
In control group two, fall ill to watermelon blight more serious, disease index is up to 39.4 (tables 1).Using having added bud
The Brevibacillus brevis wettable powder of spore germinant can significantly reduce the disease index to watermelon blight, and relative control effect reaches
79.9%；But using control group a pair of the watermelon blight for the Brevibacillus brevis wettable powder for being not added with gemma germinant
Control effect it is poor, relative control effect is only 41.37%.The short of gemma germinant is added from volume increase situation it can also be seen that applying
The watermelon cell production of bacillus brevis wettable powder reaches 288 kilograms, than control volume increase 60.9%；And gemma is not added to sprout
The Brevibacillus brevis wettable powder of agent（Control group one）Only than clear water group（Control group two）Volume increase 12.8% is tied from experiment
Fruit this it appears that added the Brevibacillus brevis wettable powder of gemma germinant have volume increase, upgrading, the effect of diseases prevention
Fruit.
1 different disposal of table is to the control effect of watermelon blight
| Processing | Disease index | Incidence | Relative control effect | Yield (kg) | Rate of growth (%) |
| Control group two | 39.4 | 64.5% | - | 179 | |
| Control group one | 23.1 | 57.9% | 41.37% | 202 | 12.8 |
| Test group | 7.9 | 17.4% | 79.9% | 288 | 60.9 |
Claims (2)
1. a kind of Brevibacillus brevis wettable powder, which is characterized in that the Brevibacillus brevis wettable powder is under
Row parts by weight form：40-70 parts of Brevibacillus brevis zymotic fluid；Filler：20-30 parts of white carbon；Wetting agent：Dodecyl sulphate
1-4 parts of sodium；Dispersant：T-shaped lignin 0.2-1 parts；Gemma germinant：0.1-0.5 parts of inosine, 0.1-0.5 parts of fructose；It is ultraviolet
Protective agent：0.5-2 parts of lecithin, 0.2-1 parts of powder-beta-dextrin；Drying protectant：1-4 parts of maltose, 0.2-1 parts of sucrose；Wherein, institute
The preparation method for the Brevibacillus brevis wettable powder stated is made of suddenly the following steps：
First, the preparation of Brevibacillus brevis zymotic fluid
Culture presevation pipe is taken out, tablet is drawn with LB solid mediums and recovers, when 30 DEG C of cultures 48 are small, the picking list under tablet
A colony inoculation is into 50 milliliters of LB culture mediums, and when 30 DEG C of shake cultures 24 are small in the incubator, seed uses 5% inoculum concentration,
The bacillus liquid culture medium being seeded in the big triangular flasks of 5L, liquid amount 2L, when 30 DEG C of shake culture 16-28 are small, detection
Its bacterial concentration, bacterium solution content are more than 6,000,000,000/milliliter, you can as Brevibacillus brevis seed liquor；By Brevibacillus brevis kind
Sub- liquid is seeded in the fermentation tank of 1000L, and liquid amount is the fermentation medium of 600L, and throughput is liquid per minute when being preceding 16 small
Gas ratio is 1：0.6, throughput is 1 thereafter：1；Open stirring, rotating speed 160r/min；It is 30 DEG C when 24 is small before cultivation temperature,
Afterwards, temperature improved 1 DEG C, when culture 28-36 is small when every 4 is small, detects its spore content more than 8,000,000,000 CFU/ml, you can terminate hair
Ferment, as Brevibacillus brevis zymotic fluid；
Wherein, the LB culture mediums：Peptone 10g, yeast extract 5g, sodium chloride 10g, water 1000mL, pH7.2, solid medium
Middle addition agar 2%；
Wherein, the bacillus liquid culture medium：Glucose 10g/L, 8 g/L of soluble starch, 15 g/L of bean cake powder, fish meal
5 g/L, 0.5 g/L of magnesium sulfate, 0.5 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, dipotassium hydrogen phosphate 0.5 g/L, pH7.2；
Wherein, the fermentation medium：Glucose 5g/L, 20 g/L of corn flour, 50 g/L of bean cake powder, 10 g/ of corn starch
L, 0.5 g/L of magnesium sulfate, 0.5 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, dipotassium hydrogen phosphate 0.5 g/L, pH7.2；
The condition of each medium sterilization is：0.10-0.15MPa, 121 DEG C sterilize 30 minutes；
2nd, by 40-70 parts of Brevibacillus brevis zymotic fluid；Filler：20-30 parts of white carbon；Uv-protector：Lecithin 0.5-2
Part, 0.2-1 parts of powder-beta-dextrin；Drying protectant：1-4 parts of maltose, 0.2-1 parts of sucrose, is spray-dried after mixing, obtains short bud
Spore bacillus pulvis；
3rd, by wetting agent：1-4 parts of lauryl sodium sulfate；Dispersant：T-shaped lignin 0.2-1 parts；Gemma germinant：Inosine
0.1-0.5 parts, 0.1-0.5 parts of fructose；It is uniformly mixed so as to obtain the auxiliary agent of Brevibacillus brevis；
4th, by step 2 and the obtained Brevibacillus brevis pulvis of step 3 and auxiliary agent mixing and excessively by airslide disintegrating mill powder
Broken 400 mesh sieve of mistake detects its spore content not less than 10,000,000,000 cfu/g, and suspensibility is not less than 85%, and wetting time is not higher than 80s,
Water content is not higher than 5%, vacuum packaging, you can obtain Brevibacillus brevis wettable powder.
2. a kind of Brevibacillus brevis wettable powder according to claim 1, which is characterized in that by following parts by weight
Composition：
60 parts of Brevibacillus brevis zymotic fluid；Filler：25 parts of white carbon；Wetting agent：3 parts of lauryl sodium sulfate；Dispersant：T
0.5 part of type lignin；Gemma germinant：0.2 part of inosine, 0.4 part of fructose；Uv-protector：1.5 parts of lecithin, powder-beta-dextrin
0.8 part；Drying protectant：3 parts of maltose, 0.6 part of sucrose.
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| CN115281216A (en) * | 2022-08-10 | 2022-11-04 | 河北省科学院生物研究所 | Application of brevibacillus brevis ZLP-151 in biological prevention and control |
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| CN103039530A (en) * | 2012-12-27 | 2013-04-17 | 陕西恒田化工有限公司 | Wettable powder of bacillus methylotrophicus and preparation method thereof |
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| CN103704273A (en) * | 2014-01-13 | 2014-04-09 | 湖南农业大学 | Endogenous brevibacillus brevis wettable powder |
| WO2014193746A1 (en) * | 2013-05-31 | 2014-12-04 | Novozymes Bioag A/S | Compositions and methods for enhancing germination |
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| CN103039530A (en) * | 2012-12-27 | 2013-04-17 | 陕西恒田化工有限公司 | Wettable powder of bacillus methylotrophicus and preparation method thereof |
| WO2014193746A1 (en) * | 2013-05-31 | 2014-12-04 | Novozymes Bioag A/S | Compositions and methods for enhancing germination |
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