CN105494441A - Preparing method for bacillus pumilus wettable powder - Google Patents
Preparing method for bacillus pumilus wettable powder Download PDFInfo
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- CN105494441A CN105494441A CN201510951106.4A CN201510951106A CN105494441A CN 105494441 A CN105494441 A CN 105494441A CN 201510951106 A CN201510951106 A CN 201510951106A CN 105494441 A CN105494441 A CN 105494441A
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- bacillus pumilus
- powder
- gemma
- parts
- wetting
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- 229960003786 inosine Drugs 0.000 description 1
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- 229960000310 isoleucine Drugs 0.000 description 1
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- 239000000787 lecithin Substances 0.000 description 1
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Classifications
-
- 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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- 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
- A01N25/12—Powders or granules
- A01N25/14—Powders or granules wettable
Landscapes
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Wood Science & Technology (AREA)
- Agronomy & Crop Science (AREA)
- Environmental Sciences (AREA)
- Toxicology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of biopesticide and microbial fertilizer, and particularly discloses bacillus pumilus wettable powder containing a spore germinating agent and a preparing method thereof. The wettable powder is prepared from, by weight, 40-70 parts of bacillus pumilus fermentation liquor, 20-30 parts of filler (bentonite), 1-4 parts of a wetting agent (polyethylene glycol), 0.2-1 part of a dispersing agent (lauryl sodium sulfate), 0.1-0.5 part of a spore germinating agent (L-alanine), 0.1-0.5 part of calcium 2,6-pyridine dicarboxylate, 0.5-2 parts of an ultraviolet protective agent (carboxymethylcellulose), 0.2-1 part of beta-dextrin, 1-4 parts of a drying protective agent (maltose) and 0.2-1 part of sodium glutamate. After the prepared bacillus pumilus wettable powder is applied to a land, spores germinate at a high speed, the germination rate is high, fertilizer efficiency and the prevention and control effect on plant diseases can be improved obviously, the usage amount of short spores is reduced, and production cost is reduced.
Description
Technical field
The present invention relates to biopesticide and microbial manure field, be specifically related to a kind of bacillus pumilus wetting powder containing short and small gemma germinant and preparation method thereof.
Background technology
China uses a large amount of chemical pesticide preparations and chemical fertilizer every year, very easily causes residue of pesticide to exceed standard, biological concentration, not only pollution of ecological environment, also affects the health of the mankind.And biopesticide side effect is little, harmless to people, be a kind of environment friendly agricultural with bright prospects, day by day become a kind of trend of global pesticide development.
Bacillus is a kind of microorganism of most study in current biopesticide and bio-fertilizer, it has broad spectrum antibiotic activity and extremely strong anti-adversity ability, its trophozyte can be grown surely in the procreation of fungal pathogen mycelium surface, effective suppression pathogen growth, therefore, occur in control disease, promote plant strain growth, improve the aspects such as crop yield and show wide application prospect.
Bacillus pumilus a kind ofly easily in soil, is separated the microorganism obtained, found by research, wheat, beet, cucumber can not only be promoted, paddy rice, the growth of the crops such as tomato, rice blast can also be prevented and treated, stem rot of cucumber, apple canker, wilt of eggplant, wheat scab, Cabbage Leaf Spot and graw mold of tomato etc.
At present, the research of the zytase to its Biocontrol Effect and generation is mainly concentrated on both at home and abroad about the research of bacillus pumilus.Xiao Aiping etc. find that bacillus pumilus has good control efficiency to rice blast, and its useful effect composition is polypeptides matter (Xiao Aiping etc., 2003).Cui Yunlong etc. find that bacillus pumilus D82 has strong antibiosis to root rotof flax, and the dull and stereotyped fungistatic effect of zymotic fluid diluted concentration less than 100 times is 100% (Cui Yunlong etc., 1995).In addition, Xu Changlun etc. also find that the optimization bacterial strain P10 of a bacillus pumilus can have the effect (Xu Changlun etc., 1994) of Yield-increasing and sugar-increasing to beet; The separation such as Zhu Guimei obtains a bacillus pumilus TW-2 and has strong inhibitory action to rice blast fungus, has higher control efficiency to rice blast.Bacillus pumilus (Bacilluspumilus) is determined to have ability (MariM, etal, 1996 of the disease funguses such as antagonism the pathogen of Botrytis cinerea, pythium spp in recent years successively; WalkerR, etal, 1998).AkhtarM.S. report that bacillus pumilus is used for chickpea root rot as bio-bacterial manure and obtains good control efficiency by (2008).Cui Yunlong etc. (1995) report bacillus pumilus D82 has extremely strong antagonism to Cochliobolus sativus.The bacillus pumilus that equality of going on a spring outing (2001) is separated has significant inhibitory action to Pyricularia oryzae.King waits quietly being separated to a bacillus pumilus AR03, and its thalline and fermentation broth on tobacco black shank bacterium have very strong antagonistic activity, has the ability of control black shank preferably in field test results display.
At present, domestic existing correlation technique of closing production bacillus pumilus series products, as: Pan Yilou etc. are in the patent No.: the preparation method disclosing a kind of Brevibacillus brevis water dispersible granules in 2012102713964, in the patent No. 2007100215091, disclose a kind of preparation of the Brevibacillus brevis TW bacterial strain for preventing and treating rice blast, said preparation to the control efficiency of rice blast between 60%-80%.Lu Yanjun etc. are separated to a bacillus pumilus A3 all has stronger antagonism to the mycelia of Valsa paulowniae and conidium, and the zymotic fluid of A3 can reach 97.8% to the sick control efficiency of Valsa paulowniae.Li Liantai etc. disclose a kind of bacillus pumilus E4 and base cultural method and are suppressing the application on vibrios in the patent No. 2013101282710.Lian Lingli etc. are separated and obtain a bacillus pumilus EN16, and it can induce tomato to the resistance of bacterial wilt.Huanghai Sea east etc. discloses the zymotechnique of a bacillus pumilus LD-b1 and is preventing and treating Cucumber Target Leaf Spot, gray mold of cucumber, stem rot of cucumber in the patent No. 2012102903742, apple canker, wilt of eggplant, wheat scab, the application on Cabbage Leaf Spot and graw mold of tomato.The refined grade of curly hair has investigated the impact of bacillus pumilus zymotic fluid on Tomato Seeds Germination and growth of seedling, result shows: bacillus pumilus zymotic fluid is to Tomato Seeds Germination, plant height and the strain of radical bud growth and seedling heavily have obvious facilitation, wherein remarkable with the effect of 50 times of dilutions, and the successful of foliage-spray is better than filling with root and seed soaking in seedling method of application.But also there is not the preparation method of the wetting powder of concrete relevant bacillus pumilus open.
Although the microbial pesticide product being main preparation with bacillus is many, its formulation has aqua, pulvis, wetting powder, water dispersible granules, but the formulation of current bacillus is based on wetting powder, although this formulation is convenient to store and transport, in production application, there is a lot of restrictive factor.As most using gemma as the unitary basis weighing active ingredient in product in the wetting powder used, if gemma can not be sprouted in time, application effect is then not good, as Wang Qian utilize bacillus subtilis TR21 to prevent and treat Brazilian any of several broadleaf plants fusarium wilt time, pre-stage test utilizes TR21 bacterial strain NB fermentation liquid irrigating root and axil inoculation effectively can prevent and treat fusarium wilt, but utilize gemma to prepare the filling root of wetting powder and axil effect undesirable, demonstrating the pure gemma after removing zymotic fluid may make preventive effect decline because sprouting difficulty.
Bacillus is widely distributed at occurring in nature, when external condition is applicable to growth, brood cell can grow into trophosome by resting state through series of complex biochemical reactions and recover metabolism, and this process is called sprouting (germination).Occurring in nature gemma sprouts the impact by exogenous nutrition material, this kind of nutriment is referred to as " germinant ", usual nutrition induction gemma sprouts the single effect comprising amino acid, carbohydrate and fast cry of certain animals nucleosides, also comprises their synergy (such as: AGFK; A:L mono-asparagus fern phthalein amine; G: glucose; F: fructose; K: potassium ion).When nutriment touches gemma, can there is a series of change in gemma inside, the inner H of (1), gemma core
tenand Zn
2+discharge, cause gemma core internal pH to rise to alkalescent by faintly acid; (2), the inner Ca of gemma core
2+--a large amount of releases of DPA; (3), the hydration of gemma core, inner Ca
2+--DPA substitute by water, the warm tolerance of gemma declines; (4), the hydrolysis of the gemma skin glycan layer of cortex, gemma swelling of nucleus; (5), the further hydration of gemma core, gemma core internal protein mobility strengthen, enzymic activity strengthen, synthesis ATP; (6), degraded SASP albumen, to discharge gemma DNA, and then guide the synthesis of RNA, protein, DNA in gemma process of growth.
After the gemma germinant mixing that gemma and promotion are sprouted within the several seconds, germinant strides across the surface of gemma clothing and cortex arrival intine fast, interact with the receptor protein be positioned on intine, gemma is excited to enter germinating, then start a succession of sprouting event, germination process afterwards can not stop because removing germinant.
In Bacillus, Bacillus subtillis, thuringiensis, Bacillus anthracis and Bacillus cereus receive much concern because of closely bound up with the productive life of the mankind.Research shows, the genome of Bacillus subtillis comprise a series of with
The gene cluster that antibiotic and essential industry are correlated with enzyme, Bacillus anthracis and Bacillus cereus all have very strong pathogenic to people and animals, because the former is for the manufacture of biological weapons, and the latter produces the toxin causing people's vomiting and diarrhoea, therefore both moment threaten the health and lives safety of the mankind, when being in spore state, they all do not possess infection ability, but after entering germinating, start to have the ability infecting people and animals, and the final toxin producing pathogenic even causing death; And the pathogenetic bacteria Su Yun gold brood cell of insect
Bacillus has special toxic action to insect, can cause septicemia after the germination in pin main body, and their brood cell effectively can improve the virulence of insecticidal crystal protein, and the sprouting of brood cell may be necessary in synergistic effect.Therefore, to the research of bacillus germination process for suppressing it pathogenic, safeguard that human health and life security have vital theory significance and actual application value, therefore these four kinds of gemma are sprouted mechanism to it and are studied often to relevant germinant, but disclose about the gemma germinant of bacillus pumilus yet there are no.The report of the research adding gemma germinant in bacillus pumilus wetting powder is not had yet.
In addition, due to different bacillus and wetting agent, the compatibility of the auxiliary agents such as dispersant, carrier is different to each series bacillus adsorptivity, cannot for the preparation of the bacillus pumilus wetting powder containing gemma germinant by the formula of existing bacillus wettable powder or preparation technology, the adsorbing capacity of effective bacterium in wetting powder, survival rate, to the control efficiency of harmful bacteria and the stability difference of wettable powder dosage larger, the preparation method of its whole wetting powder must give overall consideration to each carrier adsorption ability, to protective effect in the dry run of bacillus pumilus, wetting agent, dispersant is to the suspensibility of wetting powder, the impact of MEBO ribbon gauze, and carrying out comprehensively screening to gemma germinant, can to obtain MEBO ribbon gauze short, suspensibility is high, the gemma holding time is of a specified duration, use rear gemma energy fast-germination, the bacillus pumilus wetting powder that preventive effect is good.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, the invention provides a kind of bacillus pumilus wetting powder containing gemma germinant and preparation method thereof, after gemma germinant can make bacillus pumilus wetting powder incorporate water, short and small gemma energy fast-germination, greatly improve the germination rate of short and small gemma, its bacillus pumilus wetting powder spore content prepared is for being not less than 20,000,000,000 CFU/ gram, suspensibility is not less than 75%, wetting time is not higher than 85S, finess disintegrating crosses 400 mesh sieves, moisture is not higher than 5%, preparation method stablizes, bacillus pumilus consumption is little, cost is low.
In order to achieve the above object, the following weight fraction composition of a kind of bacillus pumilus wetting powder: bacillus pumilus zymotic fluid 40-70 part; Filler: bentonite 20-30 part; Wetting agent: polyethylene glycol 1-4 part; Dispersant: lauryl sodium sulfate 0.2-1 part; Gemma germinant: ALANINE 0.1-0.5 part, 2, dipicolimic acid 2 calcium 0.1-0.5 parts; Uv-protector: carboxymethyl cellulose 0.5-2 part, powder-beta-dextrin 0.2-1 part; Drying protectant: maltose 1-4 part, sodium glutamate 0.2-1 part.
A preparation method for bacillus pumilus wetting powder, is made up of suddenly the following steps:
One, the preparation of bacillus pumilus zymotic fluid
Take out culture presevation pipe, draw flat board with NB solid culture medium and recover, cultivate 48 hours for 30 DEG C.Under flat board in the single colony inoculation to 50 of picking milliliter NB medium, in incubator, 30 DEG C shake cultivation 24 hours.Seed adopts the inoculum concentration of 5%, and be seeded to the bacillus pumilus liquid nutrient medium in the large triangular flask of 5L, liquid amount is 2L, 16-28 hour is cultivated in 30 DEG C of concussions, detect its bacterial concentration, bacterium liquid hold-up, more than 6,000,000,000/milliliter, namely can be used as bacillus pumilus seed liquor.Be seeded in the fermentation tank of 1000L by bacillus pumilus seed liquor, liquid amount is the fermentation medium of 600L, throughput be first 16 hours for liquid-gas ratio per minute be 1:0.6, throughput is 1:1 thereafter; Open stirring, rotating speed is 160r/min, and cultivation temperature is 30 DEG C in first 24 hours, thereafter, temperature is improved 1 DEG C in every 4 hours, cultivates 28-36 hour, detects its spore content more than 20,000,000,000 CFU/ml, can terminate fermentation, as bacillus pumilus zymotic fluid;
Wherein, described NB medium: peptone 10g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, add agar 2% in solid culture medium;
Wherein, described bacillus pumilus liquid nutrient medium: glucose 10g/L, soluble starch 8g/L, analysis for soybean powder 15g/L, fish meal 5g/L, magnesium sulfate 0.5g/L, manganese sulphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, calcium carbonate 2g/L, pH7.2;
Wherein, described fermentation medium: glucose 5g/L, corn flour 25g/L, bean cake powder 70g/L, wheat bran 10g/L, fish meal 5g/L, magnesium sulfate 0.5g/L, manganese sulphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, calcium carbonate 5g/L, pH7.2;
The condition of each medium sterilization is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes;
Two, by bacillus pumilus zymotic fluid 40-70 part; Filler: bentonite 20-30 part; Uv-protector: carboxymethyl cellulose 0.5-2 part, powder-beta-dextrin 0.2-1 part; Drying protectant: maltose 1-4 part, sodium glutamate 0.2-1 part; Spraying dry after mixing, obtains bacillus pumilus pulvis;
Three, by wetting agent: polyethylene glycol 1-4 part; Dispersant: lauryl sodium sulfate 0.2-1 part; Gemma germinant: ALANINE 0.1-0.5 part, 2, dipicolimic acid 2 calcium 0.1-0.5 part mixings obtain the auxiliary agent of bacillus pumilus;
Four, bacillus pumilus pulvis step 2 and step 3 obtained and auxiliary agent mix and cross pulverized 400 mesh sieves by airslide disintegrating mill, detect its spore content and be not less than 20,000,000,000 cfu/g, suspensibility is not less than 75%, wetting time is not higher than 85s, moisture is lower than 5%, vacuum packaging, is bacillus pumilus wetting powder.
Wherein, described bacillus pumilus wetting powder optimum weight part composition: bacillus pumilus zymotic fluid 60 parts; Filler: bentonite 24 parts; Wetting agent: polyethylene glycol 2 parts; Dispersant: lauryl sodium sulfate 0.6 part; Gemma germinant: ALANINE 0.2 part, 2, dipicolimic acid 2 calcium 0.3 part; Uv-protector: carboxymethyl cellulose 1 part, powder-beta-dextrin 0.5 part; Drying protectant: maltose 2 parts, sodium glutamate 0.8 part.
Occurring in nature gemma sprouts the impact by exogenous nutrition material, this kind of nutriment is referred to as " germinant ", under normal circumstances, after the gemma germinant mixing that gemma and promotion are sprouted within the several seconds, germinant strides across the surface of gemma clothing and cortex arrival intine fast, interacts, excite gemma to enter germinating with the receptor protein be positioned on intine, then start a succession of sprouting event, germination process afterwards can not stop because removing germinant.Containing gemma germinant in wetting powder of the present invention: ALANINE, 2, dipicolimic acid 2 calcium; From glycosides propylhomoserin, ALANINE, valine, leucine, isoleucine, serine, threonine, methionine, cystine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, phenyl alanine, tyrosine, tryptophan, histidine, proline, inosine, 2,6 pyridine dicarboxyl calcium, carry out single factor experiment in fructose, and the germinant with the short and small gemma sprouting of obviously induction that combination of two test obtains.Through overtesting, bacillus pumilus wetting powder of the present invention and water carry out wetting mixing, after gemma germinant fully contacts with gemma, thus impel its fast-germination, through overtesting, after 30 minutes, gemma germination rate reaches more than 80%, and the short and small gemma not adding gemma germinant after 4 hours, its gemma germination rate only reaches about 45%.
The present inventor passes through carrier (as bentonite, talcum powder, kaolin, diatomite, white carbon, rock phosphate, precipitated calcium carbonate), auxiliary agent (wetting agent and dispersant) (polyvinyl alcohol, polyethylene glycol, CMC-Na, Tween-60, neopelex, T-shaped lignin, lauryl sodium sulfate, Tea Saponin, MorwetEFW, MorwetD425, My100, D110, Arabic power glue, 2-naphthalene sulfonic acid formaldehyde polymer sodium salt, OPEO, diisooctyl Disodium sulfosuccinate, methylcellulose, sodium lignin sulfonate) filler (diatomite, kaolin, bentonite, white carbon, precipitated calcium carbonate, talcum powder), ultraviolet protective agent (ascorbic acid, dextrin, neopelex, Congo red, carboxymethyl cellulose, lecithin), drying protectant (skim milk powder, sucrose, sodium glutamate, maltose, mannose, methylcellulose etc.) screen, it is often planted auxiliary agent and survives to the gemma of bacillus pumilus, suspension, wettability is tested, in conjunction with the gemma germinant of bacillus pumilus, the formula of bacillus pumilus wetting powder is determined finally by many wheel single factor test and orthogonal experiment.20,000,000,000 cfu/g are not less than through producing its spore content of bacillus pumilus wetting powder obtained, suspensibility is not less than 75%, wetting time is not higher than 85s, moisture is lower than 5%, by checking, bacillus pumilus wetting powder of the present invention is preserved 1 year at 20-25 DEG C, and its gemma survival rate also can reach 16,500,000,000 CFU/ gram.
Vacuum packaging adopted by wetting powder of the present invention, reduces the contact of gemma and air, reduces the probability that it is sprouted.
The moisture of wetting powder of the present invention is lower than 5%, even if containing gemma germinant (ALANINE and 2 in the wetting powder of low water content, dipicolimic acid 2 calcium), the gemma activity in its storage process is not also significantly affected, does not reduce its gemma in storage process.
The present invention compared with prior art, has the following advantages and effect:
This domesticly first the gemma germinant of bacillus pumilus is added bacillus pumilus and can carry out suitability for industrialized production by humectant, greatly improve gemma and sprout speed and germination rate, solve because gemma is sprouted slow, the control efficiency that germination rate is low and cause is poor, the problem of effect instability, reduce the consumption of short and small gemma, reduce production cost;
Available its spore content of bacillus pumilus wetting powder is not less than 20,000,000,000 cfu/g, suspensibility is not less than 75%, wetting time is not higher than 85s, moisture is lower than 5%, by checking, bacillus pumilus wetting powder of the present invention is preserved 1 year at 20-25 DEG C, and its gemma survival rate also can reach 16,500,000,000 CFU/ gram.
Embodiment
Substantive distinguishing features of the present invention can be embodied from the following examples, but these embodiments are only as illustrating, instead of limits the invention.
Embodiment 1
The following weight portion composition of a kind of bacillus pumilus wetting powder: bacillus pumilus zymotic fluid 60 parts; Filler: bentonite 24 parts; Wetting agent: polyethylene glycol 2 parts; Dispersant: lauryl sodium sulfate 0.6 part; Gemma germinant: ALANINE 0.2 part, 2, dipicolimic acid 2 calcium 0.3 part; Uv-protector: carboxymethyl cellulose 1 part, powder-beta-dextrin 0.5 part; Drying protectant: maltose 2 parts, sodium glutamate 0.8 part.
A preparation method for bacillus pumilus wetting powder, is made up of suddenly the following steps:
One, the preparation of bacillus pumilus zymotic fluid
Take out culture presevation pipe (ACCC11083 bacillus pumilus Bacilluspumilus is purchased from Chinese agriculture Microbiological Culture Collection administrative center, can prevent and treat nematodosis), draw flat board with NB solid culture medium and recover, cultivate 48 hours for 30 DEG C.Under flat board in the single colony inoculation to 50 of picking milliliter NB medium, in incubator, 30 DEG C shake cultivation 24 hours.Seed adopts the inoculum concentration of 5%, and be seeded to the bacillus pumilus liquid nutrient medium in the large triangular flask of 5L, liquid amount is 2L, 16-28 hour is cultivated in 30 DEG C of concussions, detect its bacterial concentration, bacterium liquid hold-up, more than 6,000,000,000/milliliter, namely can be used as bacillus pumilus seed liquor.Be seeded in the fermentation tank of 1000L by bacillus pumilus seed liquor, liquid amount is the fermentation medium of 600L, throughput be first 16 hours for liquid-gas ratio per minute be 1:0.6, throughput is 1:1 thereafter; Open stirring, rotating speed is 160r/min, and cultivation temperature is 30 DEG C in first 24 hours, and thereafter, temperature was improved 1 DEG C in every 4 hours, cultivate 28 hours, detecting its spore content is 21,000,000,000 CFU/ml, namely terminates fermentation, as bacillus pumilus zymotic fluid;
Wherein, described NB medium: peptone 10g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, add agar 2% in solid culture medium;
Wherein, described bacillus pumilus liquid nutrient medium: glucose 10g/L, soluble starch 8g/L, analysis for soybean powder 15g/L, fish meal 5g/L, magnesium sulfate 0.5g/L, manganese sulphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, calcium carbonate 2g/L, pH7.2;
Wherein, described fermentation medium: glucose 5g/L, corn flour 25g/L, bean cake powder 70g/L, wheat bran 10g/L, fish meal 5g/L, magnesium sulfate 0.5g/L, manganese sulphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, calcium carbonate 5g/L, pH7.2;
The condition of each medium sterilization is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes;
Two, by bacillus pumilus zymotic fluid 60 parts; Filler: bentonite 24 parts; Uv-protector: carboxymethyl cellulose 1 part, powder-beta-dextrin 0.5 part; Drying protectant: maltose 2 parts, sodium glutamate 0.8 part; Spraying dry after mixing, obtains bacillus pumilus pulvis;
Three, by wetting agent: polyethylene glycol 2 parts; Dispersant: lauryl sodium sulfate 0.6 part; Gemma germinant: ALANINE 0.2 part, 2, the 0.3 part of mixing of dipicolimic acid 2 calcium obtains the auxiliary agent of bacillus pumilus;
Four, bacillus pumilus pulvis step 2 and step 3 obtained and auxiliary agent mix and cross pulverized 400 mesh sieves by airslide disintegrating mill, detecting its spore content is 20,800,000,000 cfu/g, suspensibility is 82%, wetting time is 65s, moisture is 4.2%, vacuum packaging, is bacillus pumilus wetting powder.
The test effect of embodiment 2 bacillus pumilus wetting powder on muskmelon
Test field is positioned at Cang County village, Cangzhou, Hebei Province, selects the serious field of root-knot nematode disease to carry out.Test period is on September 25, May 6 to the same year in 2014, and test plant is muskmelon;
Select 51% (containing N17%, P2O517%, K2O17%) S-based compound fertilizer as base manure, 15d uses in advance, and average every district uses 6kg, every district is 0.2 mu, every district uses common fertilizer 30 kilograms, and muskmelon adopts conventional seeding tray seedling culture, transplants after nursery 30d; Experimental group: before transplanting, bacillus pumilus wetting powder 100 times of vacuole roots containing gemma germinant prepared by embodiment 12 hours; Transplant and spray first time with bacillus pumilus wetting powder 250 times of liquid in latter 30 days, and dilute liquid irrigating root once with bacillus pumilus wetting powder 1000 times; Control group one: before transplanting, with the bacillus pumilus wetting powder 100 times of vacuole roots 2 hours without gemma germinant; Transplant and spray first time with the bacillus pumilus wetting powder 250 times of liquid without gemma germinant in latter 30 days, and dilute liquid irrigating root once with the bacillus pumilus wetting powder 1000 times without gemma germinant; Control group two: i.e. blank group, the method operation identical with experimental group, replaces bacillus pumilus wetting powder with clear water.
Result of the test:
Output increases, and quality improves.In control group two, when only using inorganic fertilizer and common fertilizer, the morbidity of muskmelon root Root-knot is comparatively serious, and disease index reaches 64.5% (table 1).Use the disease index that the bacillus pumilus wetting powder having added gemma germinant significantly can reduce root-knot nematode, relative control effect reaches 73.5%; But the control efficiency using control group a pair root-knot nematode disease of the bacillus pumilus wetting powder not adding gemma germinant is poor, and relative control effect is only 8.8%.Also can see from volume increase situation, apply the bacillus pumilus wetting powder volume increase having added gemma germinant and reach 30%.The control group one applying the bacillus pumilus wetting powder not adding gemma germinant has only increased production 7.98% than clear water group (control group two).
Table 1 different disposal is on the impact of root knot nematode disease and preventive effect
| Process | Root knot index | Disease index | Relative control effect | Output (kg) | Rate of growth (%) |
| Control group two | 3.4 | 64.5% | - | 702 | |
| Control group one | 3.1 | 57.9% | 8.8% | 758 | 7.98 |
| Experimental group | 0.9 | 17.4% | 73.5% | 912.6 | 30 |
Claims (3)
1. a bacillus pumilus wetting powder, is characterized in that, described bacillus pumilus wetting powder is made up of following weight portion: bacillus pumilus zymotic fluid 40-70 part; Filler: bentonite 20-30 part; Wetting agent: polyethylene glycol 1-4 part; Dispersant: lauryl sodium sulfate 0.2-1 part; Gemma germinant: ALANINE 0.1-0.5 part, 2, dipicolimic acid 2 calcium 0.1-0.5 parts; Uv-protector: carboxymethyl cellulose 0.5-2 part, powder-beta-dextrin 0.2-1 part; Drying protectant: maltose 1-4 part, sodium glutamate 0.2-1 part.
2. the preparation method of a kind of bacillus pumilus wetting powder according to claim 1, is characterized in that, be suddenly made up of the following steps:
One, the preparation of bacillus pumilus zymotic fluid
Take out culture presevation pipe, draw flat board with NB solid culture medium to recover, cultivate 48 hours, under flat board in the single colony inoculation to 50 of picking milliliter NB medium for 30 DEG C, in incubator, 30 DEG C shake cultivation 24 hours, seed adopts the inoculum concentration of 5%, and be seeded to the bacillus pumilus liquid nutrient medium in the large triangular flask of 5L, liquid amount is 2L, 16-28 hour is cultivated in 30 DEG C of concussions, detect its bacterial concentration, bacterium liquid hold-up, more than 6,000,000,000/milliliter, namely can be used as bacillus pumilus seed liquor; Be seeded in the fermentation tank of 1000L by bacillus pumilus seed liquor, liquid amount is the fermentation medium of 600L, throughput be first 16 hours for liquid-gas ratio per minute be 1:0.6, throughput is 1:1 thereafter; Open stirring, rotating speed is 160r/min, and cultivation temperature is 30 DEG C in first 24 hours, thereafter, temperature is improved 1 DEG C in every 4 hours, cultivates 28-36 hour, detects its spore content more than 20,000,000,000 CFU/ml, can terminate fermentation, as bacillus pumilus zymotic fluid;
Wherein, described NB medium: peptone 10g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, add agar 2% in solid culture medium;
Wherein, described bacillus pumilus liquid nutrient medium: glucose 10g/L, soluble starch 8g/L, analysis for soybean powder 15g/L, fish meal 5g/L, magnesium sulfate 0.5g/L, manganese sulphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, calcium carbonate 2g/L, pH7.2;
Wherein, described fermentation medium: glucose 5g/L, corn flour 25g/L, bean cake powder 70g/L, wheat bran 10g/L, fish meal 5g/L, magnesium sulfate 0.5g/L, manganese sulphate 0.5g/L, potassium dihydrogen phosphate 0.5g/L, dipotassium hydrogen phosphate 0.5g/L, calcium carbonate 5g/L, pH7.2;
The condition of each medium sterilization is: 0.10-0.15MPa, 121 DEG C of sterilizings 30 minutes;
Two, by bacillus pumilus zymotic fluid 40-70 part; Filler: bentonite 20-30 part; Uv-protector: carboxymethyl cellulose 0.5-2 part, powder-beta-dextrin 0.2-1 part; Drying protectant: maltose 1-4 part, sodium glutamate 0.2-1 part; Spraying dry after mixing, obtains bacillus pumilus pulvis;
Three, by wetting agent: polyethylene glycol 1-4 part; Dispersant: lauryl sodium sulfate 0.2-1 part; Gemma germinant: ALANINE 0.1-0.5 part, 2, dipicolimic acid 2 calcium 0.1-0.5 part mixings obtain the auxiliary agent of bacillus pumilus;
Four, bacillus pumilus pulvis step 2 and step 3 obtained and auxiliary agent mix and cross pulverized 400 mesh sieves by airslide disintegrating mill, detect its spore content and be not less than 20,000,000,000 cfu/g, suspensibility is not less than 75%, wetting time is not higher than 85s, moisture is lower than 5%, vacuum packaging, is bacillus pumilus wetting powder.
3. a kind of bacillus pumilus wetting powder according to claim 1, is characterized in that, described bacillus pumilus wetting powder is made up of following weight portion: bacillus pumilus zymotic fluid 60 parts; Filler: bentonite 24 parts; Wetting agent: polyethylene glycol 2 parts; Dispersant: lauryl sodium sulfate 0.6 part; Gemma germinant: ALANINE 0.2 part, 2, dipicolimic acid 2 calcium 0.3 part; Uv-protector: carboxymethyl cellulose 1 part, powder-beta-dextrin 0.5 part; Drying protectant: maltose 2 parts, sodium glutamate 0.8 part.
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| CN110279723A (en) * | 2019-07-02 | 2019-09-27 | 李会芳 | A kind of bacillus new formulation for animals and preparation method thereof |
| CN110723920A (en) * | 2019-11-08 | 2020-01-24 | 东南大学 | Wetting agent for bacillus powder and preparation method thereof |
| CN112888775A (en) * | 2018-10-18 | 2021-06-01 | 出光兴产株式会社 | Agent for decomposing plant residue using liquid culture of Bacillus pumilus KS-C4 strain |
| WO2023227768A1 (en) * | 2022-05-27 | 2023-11-30 | Croda International Plc | Wettable powder and water dispersible granule |
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| CN102154178A (en) * | 2011-01-28 | 2011-08-17 | 广东省农业科学院植物保护研究所 | Bacillus brevis for preventing and treating hot pepper epidemic disease as well as preparation method and application of biological agent |
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