CN109880763A - A kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent - Google Patents
A kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent Download PDFInfo
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Abstract
The present invention provides a kind of preparation method and applications of high-activity biological pesticide microcapsule controlled-release agent, belong to crop disease control field.The present invention realizes the High Density Cultivation of bacillus by the method that low frequency electromagnetic field radiation treatment seed liquor, sectional material supplementing ferment, the viable count and gemma rate of bacillus are significantly improved, and microcapsule controlled-release agent is prepared by novel microcapsule technology as effective component.Compared with the traditional biological formulations of pesticide, which can keep the activity of effective component in a long time, increase the storage stability of thallus, extend the shelf life of product, the stability to disease control is significantly improved, and solves dust pollution question, it is environmentally friendly.Biological pesticide of the invention is used to prevent and treat the diseases such as soybean sclerotinia crown rot, control efficiency and conventional chemical medicament without significant difference, and no pollution to the environment, suitable industrialization production are promoted.
Description
Technical field
The invention belongs to crop disease control fields, and in particular to a kind of system of high-activity biological pesticide microcapsule controlled-release agent
Preparation Method and application.
Background technique
China is a large agricultural country, need a large amount of pesticide every year in agricultural production prevent and treat the disease of crops and
Insect pest.Chemical pesticide have many advantages, such as preventive effect is high, speed is fast, insecticidal and antibacterial spectrum is wide, at low cost, using simple, therefore obtain wide
General application.But long-term, a large amount of and Reusability of chemical pesticide, serious pollution, agriculture are brought to soil, water body and atmosphere
The remaining recall rate of pesticide is up on 90% in byproduct, also brings great hidden danger to the health of the mankind and existence.In addition,
The development cycle of novel pesticide is long, at high cost, and prevention target drug resistance gradually increases and the punching of biotechnology fast development bring
It hits, current chemical pesticide industry is made to be faced with stern challenge.Therefore, to develop pesticide industry towards the direction of health,
It must just accelerate to develop efficient, safe new pesticide, walk the road of the agricultural disease prevention and treatment of sustainable development.
Biological pesticide refers to using the intracorporal active material of biology and metabolin caused by them and manufactured can use
A kind of pesticidal preparations for carrying out the harmful organisms such as prevention and control Pest organism, compared with chemical pesticide, biological pesticide has the advantage that 1) raw
It is extensive to produce raw material sources, research and development, utilization ways are more；2) toxicity is low, degradable, environmentally safe；3) selectivity is strong, will not be to non-
Target organisms damage；4) prevention target is not likely to produce drug resistance.There are many mode classifications of biological pesticide, by effective component
Source can be divided into microbial pesticide, botanical pesticide, animal pesticide and genetically modified plants etc.；By the classification of action target
Biological insecticides, biological bactericide, biological weed killer and plant growth regulator etc. can be divided into；It can divide again by the substance to work
For living body biological pesticide and metabolite biological pesticide.Numerous studies and application at present is microbial pesticide, it is in biology
It mainly include bacterium microbe pesticide, fungal microbe pesticide, viral quasi-microorganism in occupation of maximum ratio in pesticide
Pesticide and farm antibiotics etc..The product developed mainly has Dipel, muscardine, green muscardine fungus, red stiff bacterium, trichoderma
Bacterium, jinggangmeisu, gibberellin, avermectin, kasugarnycin etc..Biological pesticide because have efficiently, wide spectrum, to safety of human and livestock and with
The features such as ecological environment is compatible meets the requirement of current China's agricultural and environment sustainable development.With mankind's environmental consciousness
Enhancing and the increase to " non-harmful product " demand, biological pesticide has broad prospects and the powerful market competitiveness, in pesticide
Middle proportion will be increasing, will become one of the hot spot of 21st century Agrochemicals.
Soybean sclerotinia crown rot is also known as white rot, be by sclerotinite (Sclerotinia sclerotiorum) caused by important disease
Evil, starts to fall ill in soybean planting to late July, and overground part of mainly causing harm, seedling stage, strain can fall ill, and the florescence is aggrieved heavy,
Generate the diseases such as seedling withered, leaf is rotten, stem rot, pod corruption.Soybean sclerotinia crown rot can occur throughout the country, and popular time disease incidence is 20-
30%, disease incidence reaches 50% or more when serious, causes soybean part or complete stool withered, and 100-grain weight degradation causes the underproduction very
To total crop failure.Currently, mostly using several chemistry such as Sukeling, thiophanate-methyl, carbendazim, dimethachlon to the chemical prevention of sclerotiniose
Pesticide, the long-term use of these medicaments be easy to cause environmental pollution, drug resistance to increase, preventive effect reduces and control cost increases.Mirror
In above-mentioned reason, sight has gradually been invested biological control by people since the 1980s.
Bacillus is a kind of aerobic bacterium being widely present in soil and nature, and bacterial metabolism is energetic,
Gemma, which can be generated, when condition changes, thallus survival is suppressed carries out self-protection.Due to its it is most of it is nontoxic, to people and animals without
Evil can secrete antibacterial protein, antibiotic, enzyme, polypeptide or lipid isoreactivity substance, and have reproduction speed is fast, resistance is strong,
The advantages that being easily colonized in plant rhizosphere becomes the plant-growth promoting rhizobacteria and plant disease biocontrol bacteria of current most study.Micro- life
Most products of material resource biological pesticide are all that must contain a considerable amount of viable bacterias in product using viable bacteria as effective component
Number can be only achieved the effect of effect, therefore viable count is the important indicator for measuring microbial pesticide quality.For bacillus
For, viable count and gemma rate are to influence the key factor of bacillus pesticidal preparations biocontrol effect, and brood-gemma and viable bacteria
Body compares the advantage strong, acidproof, alkaline-resisting, heat-resisting with resistance, and thallus can be preferably saved during modern production
Activity, easy to process, storage, transport, therefore improving by advanced fermentation technique viable count and gemma rate is in recent years
The hot spot of research.
The preparation type of the microbial pesticide of current domestic registration production is mainly wettable powder, and there are also a small amount of water
Agent, suspending agent and water dispersible granules.These dosage forms easy in inactivation in normal temperature storage, shelf life is short, therefore limits biology
The industrialized development of pesticide.Furthermore the shortcomings that wettable powder is there is also dust from flying, environment easy to pollute.Microencapsulation refers to benefit
Chemical substance or biomaterial are coated in cyst material with the method for physics, chemistry and physical chemistry or biology, and controlled
System release.Compared with above traditional formulations of pesticide, microcapsules has the characteristics that as follows: (1) can be by microorganism and external environment
Isolation, thus it is smaller by environment (light, air etc.) influence, and stability is good, and activity is high；(2) it can control Microbiological release outside
The rate of boundary's environment, achievees the effect that sustained-release and controlled release；(4) it takes water as a solvent, reduces environmental pollution.Therefore using bacillus as
It is also primary study purpose of the invention that active constituent, which is prepared into microencapsulation biological pesticide,.
Summary of the invention
Lower, the microbial source agriculture for thallus number in fermentation of bacillus production process of the existing technology and gemma rate
The problem of drug stabilisation is not high, and shelf life is short, and microorganism is easy dead inactivation, and control efficiency is caused to be deteriorated, provides a kind of high work
The preparation method and application of property biological pesticide microcapsule controlled-release agent.
To solve the above problems, the present invention uses technical solution as described below:
A kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent, which is characterized in that the preparation method includes
Following steps:
1) actication of culture: the preservation of bacteria strain of ring Bacillus cercus and bacillus amyloliquefaciens is forwarded to respectively under aseptic condition
LB slant medium is placed in 30 DEG C of constant temperature incubation 12h, and so transferring 3 times makes strain activity recovery；
2) preparation of seed liquor: Bacillus cercus and bacillus amyloliquefaciens after taking activation are connect respectively with 2% inoculum concentration
Enter in seed culture medium and carry out shaking flask culture for 24 hours, low frequency electromagnetic field radiation is applied to seed liquor intermittence during the cultivation process；It shakes
The condition of bottle culture are as follows: liquid amount 100mL/500mL, 30 DEG C of cultivation temperature, shaking speed 180rpm；
3) sectional material supplementing fermented and cultured: with 5% total inoculum concentration by Bacillus cercus seed liquor and bacillus amyloliquefaciens seed
Liquid accesses in fermentation medium, and medium pH is adjusted to 7.0-7.4, and liquid amount is the 40-50% of fermenter volume, fermentation temperature
It is 34-36 DEG C, ventilatory capacity 1.1-1.3VVM, revolving speed 220-260rpm, when bacillus grows into logarithmic phase, every
8h adds 1 supplemented medium 1, adds altogether 3 times, when bacillus grows into stationary phase, adds 1 supplemented medium
2, total fermentation time 38-40h；
4) it the preparation of micro-capsule core: after fermentation, takes fermentation liquid to be centrifuged 15min under conditions of 4 DEG C and 4000r/min, removes
Bacterial sediment is washed 2-3 times with sterile saline after supernatant, is then resuspended in 1.5% aqueous trehalose, micro-capsule is made
Core；
5) preparation of microcapsule controlled-release agent: weighing beta-cyclodextrin and be dissolved in appropriate distilled water, the heating water bath at 50 DEG C, magnetic agitation
For a period of time, until being completely dissolved to form saturated aqueous solution as micro-capsule wall material, by the micro-capsule core prepared with 1:8(V/V)
Ratio be added in above-mentioned micro-capsule wall material, then be placed in ultrasonic oscillator and carry out auxiliary cladding processing, after treatment waits for
Mixed solution is cooled to room temperature and carries out decompression suction filtration again, filter cake be washed with distilled water be placed at 40 DEG C be dried in vacuo for 24 hours to get
Biological pesticide microcapsule controlled-release agent.
In the step 2, the ingredient of seed culture medium is beef extract 5g/L, yeast extract 5g/L, peptone 10g/L, grape
Sugared 5g/L, NaCl 5g/L, K2HPO4 1.5g/L, pH are adjusted to 7.0.
In the step 2, intermittence, which applies, refers to that applying low frequency electromagnetic field every 3h radiates 1h, the ginseng of low frequency electromagnetic field
Number is frequency 60Hz, electric field strength 1000V/m, magnetic field strength 0.3mT.
In the step 3), the inoculative proportion of Bacillus cercus seed liquor and bacillus amyloliquefaciens seed liquor is 2:3.
In the step 3), the ingredient of fermentation medium is molasses 8-10g/L, cornstarch 5-7g/L, yeast extract
5.0-6.0g/L bean cake powder 5.9-6.5g/L, K2HPO41.2-1.3g/L, KH2PO40.31-0.35g/L, MnSO4·H2O
0.40-0.46g/L, NaCl 4.8-5.2 g/L, MgSO4·7H2O 0.66-0.70g/L, CaCl20.19-0.23g/L, poly- second
Enol 1.7-1.9g/L.
In the step 3), the time of logarithmic phase is the 7h to 30h of fermented and cultured, and the time of stationary phase is fermentation training
After feeding 30h.
In the step 3), the ingredient of supplemented medium 1 is molasses 8-10g/L, cornstarch 5-7g/L, yeast extract
5.0-6.0g/L, bean cake powder 5.9-6.5g/L, pH are adjusted to 7.0-7.4；The ingredient of supplemented medium 2 is K2HPO4 2.4-
2.6g/L, MnSO4·H2O 0.80-0.86g/L, NaCl 4.8-5.2g/L, CaCl20.38-0.42g/L, lanthanum chloride 0.1-
0.2g/L, pH are adjusted to 7.0-7.4, and additional amount is the 9-11% of fermentation medium volume.
In the step 5), the condition of auxiliary cladding processing is 60 DEG C of ultrasonic temperature, ultrasonic time 50min.
The application is to be used to prevent and treat soybean sclerotinia crown rot for biological pesticide microcapsule controlled-release agent of the invention, and field efficacy can
Up to 71.80%.
The method have the benefit that:
(1) present invention carries out intermittent low frequency electromagnetic to bacillus seed liquor using the biological effect of low frequency electromagnetic field radiation
Field radiation treatment, can be improved the growth metabolism rate of bacillus, it is made to have better nutrition during fermented and cultured
Material utilization, stronger growth and breeding ability are conducive to improve the thallus number and gemma rate in fermentation liquid.
(2) present invention optimizes fermentation of bacillus condition of culture, and using the method for sectional material supplementing fermentation, respectively in bud
Spore bacillus logarithmic phase and stationary phase add the supplemented medium of heterogeneity, significantly improve thallus number and gemma in fermentation liquid
Rate lays the foundation for further industrialization production.
(3) present invention uses novel microcapsule technology, and optimizes to microencapsulation, and bacillus is prepared into micro-capsule
Sustained release agent.Compared with the traditional biological formulations of pesticide, which can keep the activity of effective component in a long time, not only increase
The storage stability for adding thallus extends the shelf life of preparation, significantly improves the stability to disease control, and solves wettable
Dust pollution question existing for property pulvis, it is environmentally friendly.Biological pesticide of the invention is used for the prevention and treatment of soybean sclerotinia crown rot,
Control efficiency and conventional chemical medicament are without significant difference, and no pollution to the environment, have vast potential for future development.
Detailed description of the invention
Fig. 1: influence of the inoculum concentration to thallus number and gemma number；
Fig. 2: influence of the fermentation temperature to thallus number and gemma number；
Fig. 3: the fermentation medium pH influence to thallus number and gemma number；
Fig. 4: influence of the ratio of micro-capsule core and micro-capsule wall material to encapsulation rate；
Fig. 5: influence of the ultrasonic time to encapsulation rate.
Specific embodiment
The fermented and cultured of 1 bacillus of embodiment
1) actication of culture: respectively by ring Bacillus cercus (deposit number is ACCC BCBKL 0055) reconciliation under aseptic condition
The preservation of bacteria strain of bacillus amyloliquefaciens (deposit number is ACCC 60428) is forwarded to LB slant medium, is placed in 30 DEG C of constant temperature trainings
12h is supported, so transferring 3 times makes strain activity recovery.
2) preparation of seed liquor: Bacillus cercus and bacillus amyloliquefaciens after taking activation, respectively with 2% inoculation
(ingredient is beef extract 5g/L, yeast extract 5g/L, peptone 10g/L, glucose 5g/L, NaCl 5g/ to amount access seed culture medium
L, K2HPO4 1.5g/L, pH are adjusted to carry out shaking flask culture for 24 hours in 7.0), apply during the cultivation process to seed liquor every 3h low
Frequency electromagnetic field radiates 1h, and the parameter of low frequency electromagnetic field is frequency 60Hz, electric field strength 1000V/m, magnetic field strength 0.3mT；Shaking flask
The condition of culture are as follows: liquid amount 100mL/500mL, 30 DEG C of cultivation temperature, shaking speed 180rpm.
3) sectional material supplementing fermented and cultured: with 5% total inoculum concentration by Bacillus cercus seed liquor and bacillus amyloliquefaciens
Seed liquor access fermentation medium (ingredient be molasses 9g/L, cornstarch 6g/L, yeast extract 5.5g/L, bean cake powder 6.3g/L,
K2HPO41.25g/L KH2PO40.33g/L, MnSO4·H2O 0.43g/L, NaCl 5.0g/L, MgSO4·7H2O 0.68g/
L, CaCl20.21g/L, polyvinyl alcohol 1.8g/L) in, the two inoculative proportion is 2:3, and medium pH is adjusted to 7.2, is fermented canned
Liquid measure is 45%, and fermentation temperature is 35 DEG C, ventilatory capacity 1.2VVM, revolving speed 240rpm, grows into logarithmic phase in bacillus
That is fermented and cultured 7h starts, and adding 1 supplemented medium 1(ingredient every 8h is molasses 9g/L, cornstarch 6g/L, yeast leaching
7.2) powder 5.5g/L, bean cake powder 6.3g/L, pH are adjusted to, add altogether 3 times, grow into stationary phase in bacillus and ferment
Culture 30h starts, and adding 1 supplemented medium 2(ingredient is K2HPO42.5g/L, MnSO4·H2O 0.83g/L, NaCl
5.0g/L, lanthanum chloride 0.15g/L, CaCl27.2) 0.40g/L, pH are adjusted to, additional amount is the 10% of fermentation medium volume,
Total fermentation time is 39h.
The detection of thallus number and gemma number
A) thallus number: taking fermentation liquid to carry out gradient dilution with sterile water, counts thallus number using plate count；B) gemma number:
Fermentation liquid water bath processing 15min under lethal temperature (75-80 DEG C) is taken, gradient dilution is carried out with sterile water after cooling rapidly, adopts
Gemma number is counted with plate count；Gemma rate (%)=gemma number/thallus number × 100%.
The optimization of 2 fermentation culture conditions of embodiment
1) influence of the inoculum concentration to thallus number and gemma number: set 1% for total inoculum concentration of seed liquor in embodiment 1 respectively,
3%, 5%, 7%, other conditions are identical, take fermentation liquid to carry out the detection of thallus number and gemma number after fermentation, as a result such as Fig. 1 institute
Show.Inoculum concentration increases, thallus can fast-growth simultaneously form gemma, but when inoculum concentration exceeds a certain range, due between microorganism
The nutritional ingredient for competing living space and culture medium, while generating excessive metabolic waste, thus influence thallus continue proliferation with
And the further conversion of gemma.From fig. 1, it can be seen that its thallus number and gemma number highest when inoculum concentration is 5%, are higher or lower than 5%
When thallus number and gemma number declined, be optimum inoculation amount with 5% therefore.
2) influence of the fermentation temperature to thallus number and gemma number: set 25 for fermentation temperature in embodiment 1 respectively, 30,
35,40 DEG C, other conditions are identical, take fermentation liquid to carry out the detection of thallus number and gemma number after fermentation, as a result such as Fig. 2 institute
Show.Fermentation temperature be influence microorganism growth an important factor for one of, the height of temperature in fermentation process enzyme reaction speed,
Solubility and delivery rate of the oxygen in culture solution etc. is closely related, to influence thalli growth.As can be seen from Figure 2, fermentation temperature
Its thallus number and gemma number highest when being 35 DEG C, thallus number and gemma number are declined when higher or lower than 35 DEG C, therefore, with
35 DEG C are optimum fermentation temp.
3) influence of the fermentation medium pH to thallus number and gemma number: fermentation medium pH in embodiment 1 is arranged respectively
It is 6.8,7.2,7.6,8.0, other conditions are identical, and fermentation liquid is taken to carry out the detection of thallus number and gemma number, knot after fermentation
Fruit is as shown in Figure 3.PH can influence the degree of ionization of nutriment in culture medium, to influence microorganism to nutriment
One of an important factor for absorbing, being microorganism growth.As can be seen from Figure 3, its thallus number and gemma number when fermentation medium pH is 7.2
Highest, thallus number and gemma number are declined when higher or lower than 7.2, therefore, with 7.2 for Optimal compositions of fermentation medium pH.
Comparative example 1
1) actication of culture: in the same manner as in Example 1；
2) preparation of seed liquor: Bacillus cercus and bacillus amyloliquefaciens after taking activation are connect respectively with 2% inoculum concentration
Enter in seed culture medium (ingredient is in the same manner as in Example 1) and carries out shaking flask culture for 24 hours, the condition of shaking flask culture are as follows: liquid amount
100mL/500mL, 30 DEG C of cultivation temperature, shaking speed 180rpm；
3) sectional material supplementing fermented and cultured: in the same manner as in Example 1；
It takes fermentation liquid to carry out the detection of thallus number and gemma number after fermentation, and is compared with embodiment 1, compare low-frequency electrical
Influence of the magnetic radiation to thallus number and gemma number, the results are shown in Table 1.As known from Table 1, the method according to the invention is to seed liquor
Intermittence applies low frequency electromagnetic field radiation, and then inoculation carries out fermented and cultured, so that thallus number is from the 4.37 × 10 of comparative example 111
CFU/mL is improved to the 5.15 × 10 of embodiment 111CFU/mL, gemma rate are improved from 85.58% to 94.17%.Low frequency electromagnetic field is
Refer to that frequency is lower, is typically in the electromagnetic field of 0 ~ 300Hz, with electrotechniical development, electricity consumption facility is increasing, therefore
Low frequency electromagnetic field is widely distributed in modernized society.In test we have found that the biology radiated using low frequency electromagnetic field
Effect is learned, intermittent low frequency electromagnetic field radiation treatment is carried out to bacillus seed liquor, can be improved the growth generation of bacillus
It thanks to rate, makes it that there is better utilization of nutrients, stronger growth and breeding ability, identical during fermented and cultured
Fermentation time in can obtain more thallus numbers and gemma number.
Comparative example 2
1) actication of culture: in the same manner as in Example 1；
2) preparation of seed liquor: in the same manner as in Example 1；
3) fermented and cultured: Bacillus cercus seed liquor and the access of bacillus amyloliquefaciens seed liquor are sent out with 5% total inoculum concentration
In ferment culture medium (ingredient is same as Example 1), the two inoculative proportion is 2:3, and medium pH is adjusted to 7.2, and ferment canned liquid
Amount is 45%, and fermentation temperature is 35 DEG C, ventilatory capacity 1.2VVM, revolving speed 240rpm, total fermentation time 39h.
It takes fermentation liquid to carry out the detection of thallus number and gemma number after fermentation, and is compared with embodiment 1, it is relatively low
Influence of the frequency electromagnetic radiation to thallus number and gemma number, the results are shown in Table 2.As known from Table 2, the method according to the invention carries out
Sectional material supplementing fermented and cultured, so that thallus number is from the 3.92 × 10 of normal fermentation culture11CFU/mL improves to 5.15 ×
1011CFU/mL, gemma rate are improved from 79.852% to 94.17%, promote significant effect.The present invention uses sectional material supplementing fermentation side
Method is mainly carbon source and nitrogen source in the supplemented medium 1 that bacillus logarithmic phase is added, it is possible to reduce the feedback of high concentration substrate
Inhibit, optimizes biomass growth rate, to improve thallus number；It is mainly some inorganic in the supplemented medium 2 that stationary phase is added
Salt ion and lanthanum chloride, can be improved the conversion ratio of gemma, to improve gemma number.
The preparation of 3 microcapsule controlled-release agent of embodiment
1) preparation of micro-capsule core: the fermentation liquid in Example 1 is centrifuged 15min under conditions of 4 DEG C and 4000r/min, removes
Bacterial sediment is washed 2-3 times with sterile saline after supernatant, is then resuspended in 1.5% aqueous trehalose, micro-capsule is made
Core.
2) preparation of microcapsule controlled-release agent: weighing beta-cyclodextrin and be dissolved in appropriate distilled water, the heating water bath at 50 DEG C, magnetic force
Stirring a period of time, until being completely dissolved to form saturated aqueous solution as micro-capsule wall material, by the micro-capsule core prepared with 1:8
(V/V) ratio is added in above-mentioned micro-capsule wall material, then is placed in ultrasonic oscillator and carries out auxiliary cladding processing, and condition is super
Sound temperature 60 C, ultrasonic time 50min, after treatment solution to be mixed are cooled to room temperature and carry out decompression suction filtration again, and filter cake is used
Distillation water washing, which is placed at 40 DEG C, to be dried in vacuo for 24 hours to get microcapsule controlled-release agent.
The optimization of 4 micro-capsule preparation condition of embodiment
1) influence of the ratio of micro-capsule core and micro-capsule wall material to encapsulation rate: respectively by micro-capsule core in embodiment 3 and micro-capsule wall material
The ratio setting of material is 1:4,1:6,1:8,1:10, and other conditions are constant, carries out encapsulation rate detection after preparing microcapsule controlled-release agent,
Thallus number/investment thallus number × 100% in encapsulation rate=micro-capsule, as a result as shown in Figure 4.As can be seen from Figure 4, when micro-capsule core and micro-capsule wall
In 1:4-1:8, encapsulation rate is continuously increased the ratio of material, reaches maximum value in 1:8, in 1:10 encapsulation rate under
Drop.The dosage of micro-capsule wall material is more, and covered effect is better, and encapsulation rate is higher, but when micro-capsule wall material uses excessive, due to
Reaction is reversible reaction, so that cladding process is suppressed reduces encapsulation rate instead, therefore with 1:8 for best micro-capsule core and micro-capsule
The ratio of wall material.
2) influence of the ultrasonic time to encapsulation rate: setting 30,40,50,60min for ultrasonic time in embodiment 3 respectively,
Other conditions are constant, carry out encapsulation rate detection, thallus number/investment thallus number in encapsulation rate=micro-capsule after preparing microcapsule controlled-release agent
× 100%, as a result as shown in Figure 5.As shown in Figure 5, when ultrasonic time is in 30-50min, encapsulation rate is continuously increased,
Reach maximum value when 50min, encapsulation rate is declined in 60min, therefore with 50min for best ultrasonic time.
5 microcapsule controlled-release agent storage stability test of embodiment
Using microcapsule controlled-release agent made from embodiment 3 as test group 1, with bacillus amyloliquefaciens wettable powder (effective component
Content: 1,000,000,000/gram, producer: first agriculture biology) it is used as test group 2, with bacillus subtilis aqua (active constituent content: 200,000,000/milli
Rise, producer: labor gram agricultural) it is used as test group 3, while storing at room temperature 12 months, detection thallus survival in every 3 months
Rate, the results are shown in Table 3.As known from Table 3, during the microcapsule controlled-release agent of test group 1 stores at room temperature, thallus survival rate becomes
Change less, when by 12nd month, thallus survival rate remains to reach 93.25%, be significantly higher than test group 2 wettable powder and
The aqua of test group 3 illustrates that microcapsule controlled-release agent of the invention can effectively keep the activity of thallus, improves the stable storing of preparation
Property, extend the shelf life of preparation.
6 soybean sclerotinia crown rot field controling test of embodiment
Test is located at Jiangsu Province, Guanyun County, the Lianyungang farm Yang Qiao soybean planting base, with microcapsule controlled-release made from embodiment 3
Agent is as test group 1, using 50% Sukeling wettable powder as test group 2, using 40% dimethachlon wettable powder as test
Group 3 as a control group with clear water, carries out soybean sclerotinia crown rot field control using 80% carbendazol wettable powder as test group 4
Test.4 test groups and 1 control group, every group is repeated 3 times, totally 15 cells, and about 100 plants of soybean of every cell, cell is arranged at random
Column, surrounding stay protection to go.Spray pesticide is carried out to cauline leaf in soybean florescence early stage, is administered 1 time every 7d, is administered 3 times altogether.
7d investigation diseased plant number and disease index, calculate control efficiency, the results are shown in Table 4 after the 3rd application.As known from Table 4, it tests
The control efficiency of 1 pair of soybean sclerotinia crown rot of group reaches 71.80%, does not have significance difference with the control efficiency of other chemical agent test groups
It is different, illustrate that the harm and development of soybean sclerotinia crown rot, and said preparation can be effectively controlled in biological pesticide microcapsule controlled-release agent of the invention
No pollution to the environment is suitable for promoting the use of.
Severity Scaling standard: 0 grade, cauline leaf disease-free spot；1 grade, cauline leaf begins to show fragmentary small scab；2 grades, scab accounts for cauline leaf 1/4
Below；3 grades, scab accounts for cauline leaf 1/4-1/2；4 grades, scab accounts for 1/2 or more cauline leaf.
Disease index=[Σ (diseased plant numbers at different levels × corresponding series)/(investigation total strain number × highest series)] × 100%
Control efficiency=[(control group disease index-test group disease index)/control group disease index] × 100%.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by the limit of embodiment
System, other any changes made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be
Equivalence replacement mode, is included within the scope of the present invention.
Claims (9)
1. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent, which is characterized in that the preparation method packet
Include following steps:
1) actication of culture: the preservation of bacteria strain of ring Bacillus cercus and bacillus amyloliquefaciens is forwarded to respectively under aseptic condition
LB slant medium is placed in 30 DEG C of constant temperature incubation 12h, and so transferring 3 times makes strain activity recovery；
2) preparation of seed liquor: Bacillus cercus and bacillus amyloliquefaciens after taking activation are connect respectively with 2% inoculum concentration
Enter in seed culture medium and carry out shaking flask culture for 24 hours, low frequency electromagnetic field radiation is applied to seed liquor intermittence during the cultivation process；It shakes
The condition of bottle culture are as follows: liquid amount 100mL/500mL, 30 DEG C of cultivation temperature, shaking speed 180rpm；
3) sectional material supplementing fermented and cultured: with 5% total inoculum concentration by Bacillus cercus seed liquor and bacillus amyloliquefaciens seed
Liquid accesses in fermentation medium, and medium pH is adjusted to 7.0-7.4, and liquid amount is the 40-50% of fermenter volume, fermentation temperature
It is 34-36 DEG C, ventilatory capacity 1.1-1.3VVM, revolving speed 220-260rpm, when bacillus grows into logarithmic phase, every
8h adds 1 supplemented medium 1, adds altogether 3 times, when bacillus grows into stationary phase, adds 1 supplemented medium
2, total fermentation time 38-40h；
4) it the preparation of micro-capsule core: after fermentation, takes fermentation liquid to be centrifuged 15min under conditions of 4 DEG C and 4000r/min, removes
Bacterial sediment is washed 2-3 times with sterile saline after supernatant, is then resuspended in 1.5% aqueous trehalose, micro-capsule is made
Core；
5) preparation of microcapsule controlled-release agent: weighing beta-cyclodextrin and be dissolved in appropriate distilled water, the heating water bath at 50 DEG C, magnetic agitation
For a period of time, until being completely dissolved to form saturated aqueous solution as micro-capsule wall material, by the micro-capsule core prepared with 1:8(V/V)
Ratio be added in above-mentioned micro-capsule wall material, then be placed in ultrasonic oscillator and carry out auxiliary cladding processing, after treatment waits for
Mixed solution is cooled to room temperature and carries out decompression suction filtration again, filter cake be washed with distilled water be placed at 40 DEG C be dried in vacuo for 24 hours to get
Biological pesticide microcapsule controlled-release agent.
2. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 2, the ingredient of seed culture medium is beef extract 5g/L, yeast extract 5g/L, peptone 10g/L, glucose
5g/L, NaCl 5g/L, K2HPO4 1.5g/L, pH are adjusted to 7.0.
3. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 2, intermittence, which applies, refers to that applying low frequency electromagnetic field every 3h radiates 1h, and the parameter of low frequency electromagnetic field is
Frequency 60Hz, electric field strength 1000V/m, magnetic field strength 0.3mT.
4. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 3), the inoculative proportion of Bacillus cercus seed liquor and bacillus amyloliquefaciens seed liquor is 2:3.
5. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 3), the ingredient of fermentation medium is molasses 8-10g/L, cornstarch 5-7g/L, yeast extract 5.0-
6.0g/L, bean cake powder 5.9-6.5g/L, K2HPO41.2-1.3g/L, KH2PO40.31-0.35g/L, MnSO4·H2O 0.40-
0.46g/L, NaCl 4.8-5.2 g/L, MgSO4·7H2O 0.66-0.70g/L, CaCl20.19-0.23g/L, polyvinyl alcohol
1.7-1.9g/L。
6. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 3), the time of logarithmic phase is the 7h to 30h of fermented and cultured, and the time of stationary phase is fermented and cultured
30h after.
7. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 3), the ingredient of supplemented medium 1 is molasses 8-10g/L, cornstarch 5-7g/L, yeast extract 5.0-
6.0g/L, bean cake powder 5.9-6.5g/L, pH are adjusted to 7.0-7.4；The ingredient of supplemented medium 2 is K2HPO42.4-2.6g/L
MnSO4·H2O 0.80-0.86g/L, NaCl 4.8-5.2g/L, CaCl20.38-0.42g/L, lanthanum chloride 0.1-0.2g/L, pH
It is adjusted to 7.0-7.4, additional amount is the 9-11% of fermentation medium volume.
8. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent according to claim 1, feature
It is, in the step 5), the condition of auxiliary cladding processing is 60 DEG C of ultrasonic temperature, ultrasonic time 50min.
9. a kind of preparation method and application of high-activity biological pesticide microcapsule controlled-release agent described in -8 according to claim 1, special
Sign is that the application is to be used to prevent and treat soybean sclerotinia crown rot for biological pesticide microcapsule controlled-release agent of the invention, and field efficacy can
Up to 71.80%.
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| WO2021102380A1 (en) * | 2019-11-22 | 2021-05-27 | Bayer Cropscience Lp | Methods of fermentation of recombinant bacillus spores |
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| CN104894010A (en) * | 2015-05-18 | 2015-09-09 | 青岛根源生物技术集团有限公司 | Compound microbial fertilizer for antagonism of soil-borne fungal diseases, and preparation method and application thereof |
| US20180049443A1 (en) * | 2015-03-27 | 2018-02-22 | Industrias Químicas Del Vallés, S.A. | A strain of bacillus amyloliquefaciens and its use in the control of diseases caused by bacteria and fungi in plants |
| CN108513988A (en) * | 2018-03-27 | 2018-09-11 | 湖南省蓝正微生物科技开发有限公司 | Microorganism composite pesticide |
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| US20180049443A1 (en) * | 2015-03-27 | 2018-02-22 | Industrias Químicas Del Vallés, S.A. | A strain of bacillus amyloliquefaciens and its use in the control of diseases caused by bacteria and fungi in plants |
| CN104894010A (en) * | 2015-05-18 | 2015-09-09 | 青岛根源生物技术集团有限公司 | Compound microbial fertilizer for antagonism of soil-borne fungal diseases, and preparation method and application thereof |
| CN108513988A (en) * | 2018-03-27 | 2018-09-11 | 湖南省蓝正微生物科技开发有限公司 | Microorganism composite pesticide |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021102380A1 (en) * | 2019-11-22 | 2021-05-27 | Bayer Cropscience Lp | Methods of fermentation of recombinant bacillus spores |
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Application publication date: 20190614 |