CN105886435A - Complex microorganism bacterial agent with biotic resistance and film forming property as well as preparation method and application of complex microorganism bacterial agent - Google Patents
Complex microorganism bacterial agent with biotic resistance and film forming property as well as preparation method and application of complex microorganism bacterial agent Download PDFInfo
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- CN105886435A CN105886435A CN201610305515.1A CN201610305515A CN105886435A CN 105886435 A CN105886435 A CN 105886435A CN 201610305515 A CN201610305515 A CN 201610305515A CN 105886435 A CN105886435 A CN 105886435A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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Abstract
The invention provides a method for preparing a complex microorganism bacterial agent with biotic resistance and a film forming property. The method comprises the following steps: (1) culturing bacillus subtilis, bacillus amyloliquefaciens and phyllobacterium sp respectively to obtain respective suspension solvents; (2) uniformly mixing the suspension solvents of all strains to obtain a mixed strain suspension solvent; (3) adjusting the viscosity of the mixed strain suspension solvent to be more than or equal to 2000mPa.s, so as to obtain the complex microorganism bacterial agent. The invention further provides the complex microorganism bacterial agent prepared by the method and application of the complex microorganism bacterial agent to protection of fruits. The problems of resource wastes, environment pollution and the like, which are caused by bagging in a fruit production process, are solved; meanwhile, the dosage of pesticides is greatly reduced by remarkable biological control effect and a novel, environment-friendly, safe and efficient fruit industry production mode is provided for developing green agriculture.
Description
Technical field
The present invention relates to belong to field of biological control, be specifically related to a kind of there is answering of biotic resistance and film property
Close microbial bacterial agent and preparation method thereof and the application in fruit protects.
Background technology
In modern fruit industry, the crop failure caused by disease infection and be excessively used chemical pesticide and bring
Ecological deterioration and the problem such as food safety crisis be harm human health, the weight of restriction agricultural sustainable development
Want obstacle.Wherein, the preventing and treating to fruit disease is key problem.
Currently, domestic mostly employing sprays chemical pesticide and carrys out controlling disease to technical methods such as fruit baggings.
But these methods still suffer from following point: although chemical pesticide can effectively suppress the infection of pathogenic bacteria, but for a long time,
Excess use not only can to environment, crop surface residual pesticide also can threaten food safety and
Infringement human health.Additionally, life-time service is also easy to cause fungus to develop immunity to drugs and bring to agricultural production
Many new difficult problems.For reducing pesticide directly polluting and protecting fruit from extraneous various factors fruit
Damage, bagging technique based on physical control is widely used method in current apple production.But the method
There is also a lot of shortcoming: first, bagging can cause fruit cannot accept illumination at trophophase, makes sugar etc. have
Machine matter synthetic quantity reduces, and then has influence on mouthfeel and the quality of fruit, reduces its commodity value.Next, by
In current bagging, going the operation of bag to use manually to carry out, this had both increased artificial demand, too increased more
Production cost.Additionally, due to bagging cannot Reusability, all do burning disposal with rear, cause huge money
Source waste and environmental pollution.Therefore, find a kind of safely, effectively with economical and practical method so that it is can
Effectively controlling disease, ensure that fruit safely, improve fruit quality and reduce production cost, it has also become green
Problem demanding prompt solution in color efficient fruit industry process of sustainable development.
Summary of the invention
For overcoming the problems referred to above, it is an object of the invention to provide and a kind of there is the compound of biotic resistance and film property
Microbial bacterial agent and preparation method thereof and the application in fruit protects.Described complex micro organism fungicide is sprayed
On fruit, the method that tradition bagging can be substituted, it is possible to controlling disease effectively, ensure that fruit is pacified
Entirely, fruit quality is improved.
The principle of the present invention is: in the studying for a long period of time of the Major Diseases to the fruit underproduction, the inventors discovered that
Can efficiently suppress various fungal disease biocontrol microorganisms and can the product sugar bacterium of extracellular polysaccharide.The present inventor
Obtained biocontrol microorganisms and product sugar bacterium are carried out science and compound and make complex micro organism fungicide such that it is able to effectively sends out
Wave their cooperative effect.Described complex micro organism fungicide can metabolism produce various biological alive during the fermentation
Property material.These materials, while the infecting and strengthens crop resistance of effectively suppression pathogenic bacteria, also have well
Film property, breathability and ductility, be sprayed on fruit surface can be formed in fruit face rapidly one layer thin
Semipermeable membrane, can fix complex microorganism antagonism bacterium solution, and fruit surface can be prevented again impaired；Meanwhile, it is saturating
Gas makes fruit can normally receive illumination and breathing, on fruit growth and quality without impact.Therefore, use
The present invention i.e. can reach safe and effective prevention target.
The present invention provides in first aspect has the composite microbial bacteria of biotic resistance and film property a kind of preparation
The method of agent, described method includes: (1) is cultivated bacillus subtilis (Bacillus substils) respectively, solved
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and leaf bacillus (Phyllobacterium sp), obtain
Obtain bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension；(2) by described hay
Bacillus cereus suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension mix homogeneously, thus obtain mixed vaccine
Suspension；
(3) it is more than or equal to 2000mPa s by the viscosity adjustment of described plastc ring, such as 2000
To 3000mPa s, thus obtain described complex micro organism fungicide.
The present invention provides the complex microorganism prepared by the method described in first aspect present invention in second aspect
Microbial inoculum.
The complex micro organism fungicide that the present invention provides described in second aspect present invention in the third aspect is protected at fruit
Application in protecting.
The present invention has the advantage that
1, prevention effect is good, security performance is high
The complex micro organism fungicide that the present invention provides has biotic resistance and film property, and has safety, nothing
Malicious is specific, both can ensure prevention effect when applying fruit protection, and ensure that the safety of fruit.
2, fruit quality better, yield height
Complex micro organism fungicide of the present invention has that output of sugar is high, produce the sugar cycle short, good film-forming property and property
Can stably wait feature and advantage, and there is after film forming fine air permeability and ductility, not only can rapid film formation
With the most fixing resisting substance, again can be at fruit surface formation protective layer to prevent fruit surface impaired, meanwhile,
Its breathability makes fruit can normally receive illumination and breathing, is favorably improved crop yield and quality.
3, safety non-toxic is pollution-free, sustainable development
The nontoxic edible of biotic resistance membrane safety that the complex micro organism fungicide of the present invention is formed, both will not be dirty
Dye environment, also without compromising on health.The use of this invention can effectively preserve the ecological environment, ensure food
Safety,.
4, production cost is reduced
Complex micro organism fungicide of the present invention is prone to field operation, the whole operating process demand to manpower
Measure minimum, thus make production cost be greatly lowered.
5, the foundation of the ecological agriculture is contributed to
The biomembranous performance formed by complex micro organism fungicide of the present invention is the most stable, uses it for
The Biological control of fruit, both can effective controlling disease, ensure that the quality and yield of crop.The present invention's
Promotion and application contribute to setting up brand-new fruit industry production model safe and environment-friendly, green, efficient, and can push away
The sustainable development of dynamic ecological fruit industry.
Accompanying drawing explanation
Fig. 1 fruit tree growth situation (left side is experimental group, and right side is bagging matched group)；With
Fig. 2 fruit apparent (left side is experimental group, and right side is bagging matched group).
Detailed description of the invention
As described above, the present invention provides in first aspect and has biotic resistance and film property a kind of preparation
The method of complex micro organism fungicide, described method includes:
(1) bacillus subtilis (Bacillus substils), bacillus amyloliquefaciens (Bacillus are cultivated respectively
Amyloliquefaciens) and leaf bacillus (Phyllobacterium sp), it is thus achieved that bacillus subtilis bacteria suspension, solution
Bacillus amyloliquefaciens suspension and leaf bacillus suspension；
(2) by the mixing of described bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension all
Even, thus obtain plastc ring；
(3) it is more than or equal to 2000mPa s by the viscosity adjustment of described plastc ring, thus obtains
Described complex micro organism fungicide.
Bacillus subtilis is the type culture of bacillus, it can metabolism produce protease, lipase,
The various active enzymes such as amylase, use it for the preparation of animal feed, can effectively facilitate feedstuff Middle nutrition element
Degraded, improves its nutrition absorption rate；Additionally, it can produce iturin, surface in growth course
The antibacterial material such as active extract, polymyxin, the disease control using it for crop can effectively suppress all kinds of bacterium
The activity of former bacterium and strengthen the system of crop can induction of resistance.
Bacillus amyloliquefaciens is a kind of same bacillus subtilis sibship and close bacterial strain thereof, its distribution
Extensively, easily cultivations, strong stress resistance, breeding fast and can be at the growth course a large amount of metabolites of generation, the latter is to carefully
Bacterium, fungus have a wide spectrum and efficient inhibitory action.Therefore, bacillus amyloliquefaciens is dived as great application
The biocontrol bacterial strain of power has become the study hotspot of current biological controlling plant diseases.
Leaf bacillus is a kind of aerobic gram negative bacteria, can form sugar quilt, by it by the metabolism various polysaccharide of generation
Preparation for resistance film can rapid film formation；Additionally, this bacterium does not hydrolyze starch, pectin and cellulose, therefore should
The use of bacterium will not destroy fruit quality.
It is a discovery of the invention that these three bacterium is prepared as complex microbial inoculum after science is compounding.Gained microbial inoculum
Have that output of sugar is high, produce sugar cycle feature and the advantage such as short, good film-forming property and stable performance, and after film forming
There is fine air permeability and ductility, not only can fix resisting substance with effective by rapid film formation, again can be at fruit
Surface forms protective layer to prevent fruit surface impaired.
Strain used herein is known strain, can be obtained by conventional screening, commercial means or other approach
?.
In one embodiment, can be by being either commercially available strain as herein described, such as hay spore
Bacillus can be bacillus subtilis (Bacillus subtilis), selectively, and can be from the Xi'an auspicious biotechnology of purple
Company limited (address: eastern section, Mei Wu main road, new district, riverfront, Mei County, Shaanxi Province, postcode: 722300) obtains above-mentioned bacterium
Strain.
In one embodiment, starch bud can such as be solved by being either commercially available strain as herein described
Spore bacillus can be bacillus amyloliquefaciens (Bacillus amyloliquefaciens), selectively, and can be from west
An Zirui bio tech ltd (address: eastern section, Mei Wu main road, new district, riverfront, Mei County, Shaanxi Province, postcode:
722300) above-mentioned bacterial strains is obtained.
In one embodiment, can be by being either commercially available strain as herein described, such as leaf bacillus can
To be leaf bacillus (Phyllobacterium sp), selectively, can be from Zi Rui bio tech ltd, Xi'an (
Location: eastern section, Mei Wu main road, new district, riverfront, Mei County, Shaanxi Province, postcode: 722300) obtain above-mentioned bacterial strains.
In some embodiments, in step (1), by described bacillus subtilis, described solution starch bud
Spore bacillus and described leaf bacillus the cultivation temperature of 35 DEG C carry out the most successively the Tube propagation of 72 hours, 72
Hour shake-flask culture, the seed tank culture of 72 hours and the fermentor cultivation of 72 hours, thus respectively obtain
Described bacillus subtilis bacteria suspension, described bacillus amyloliquefaciens suspension and described leaf bacillus suspension.
In some embodiments, described bacillus subtilis bacteria suspension, described bacillus amyloliquefaciens suspension and
The living bacteria count of described leaf bacillus suspension is more than or equal to 109CFU/mL, be greater than or etc.
In 1X109CFU/mL、5X109CFU/mL、1X1010CFU/mL or 5X109CFU/mL。
In some embodiments, the living bacteria count of described complex micro organism fungicide is more than or equal to 109
CFU/mL, is greater than or equal to 1X109CFU/mL、5X109CFU/mL、1X1010CFU/mL
Or 5X109CFU/mL。
In some embodiments, in step (2), described bacillus subtilis bacteria suspension, solution starch spore
The volume ratio of bacillus suspension and leaf bacillus suspension is 1:1:2～1:1:5, and optimal proportion is 1:1:4.
In some embodiments, calcium chloride solution and glycerite is used to regulate viscosity, preferred at some
Embodiment in, described in be 1% calcium chloride solution (w/v) and/or described in be the glycerol of 1% (w/v)
Solution.
The present invention provides the complex microorganism prepared by the method described in first aspect present invention in second aspect
Microbial inoculum.In some embodiments, described microbial inoculum institute after 30 DEG C of thickness film forming 30 minutes with 0.5mm
Prepare film have 2Mpa to 3.6Mpa (for example, 2,2.5,3,3.6MPa) viscosity and more than or
Person's percentage elongation (being greater than or be equal to the percentage elongation of 1.2,1.4,1.6,1.8 or 2.0) equal to 1.2%.
The present invention the third aspect provide first aspect present invention prepare described complex micro organism fungicide or
The application in fruit is protected of the complex micro organism fungicide described in second aspect present invention.
The method of application of described complex micro organism fungicide is had no particular limits by the present invention, but excellent at some
In the embodiment of choosing, the mode sprayed is used to use.In some preferred embodiments, described multiple
Close microbial bacterial agent to spray in fruit surface with the amount of 20,000 grams/acre in the setting phase.In some embodiments,
Described fruit selects free Fructus Mali pumilae and the group of Fructus actinidiae chinensis composition.
Embodiment
The following is the present invention is embodied as example, so that the present invention is described in further detail, but the present invention
Embodiment be not limited to this.
The bacillus subtilis (Bacillus substils) that in embodiment is, bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) and leaf bacillus (Phyllobacterium sp) from Xi'an the purple limited public affairs of auspicious biotechnology
Department buys and obtains.Used medium be beef extract-peptone fluid medium (5g Carnis Bovis seu Bubali cream, 10g peptone,
5g NaCl, 15g agar, 1000mL water).
Embodiment 1: the preparation of complex micro organism fungicide
(1) preparation of single bacterium bacteria suspension
Bacillus subtilis, bacillus amyloliquefaciens and leaf bacillus are cultivated the most as follows at 35 DEG C:
Tube propagation-shake-flask culture-fermentor cultivation, used medium is beef extract-peptone fluid medium (5
G Carnis Bovis seu Bubali cream, 10g peptone, 5g NaCl, 15g agar, 1000mL water), incubation time is 72h,
Finally respectively obtaining living bacteria count is 109The bacillus subtilis bacteria suspension of more than CFU/mL, solution starch bud
Spore bacillus bacteria suspension and leaf bacillus bacteria suspension.
(2) preparation of plastc ring
By the described bacillus subtilis bacteria suspension of fermentor cultivation gained, bacillus amyloliquefaciens bacteria suspension and
Leaf bacillus bacteria suspension mixes with the volume ratio of 1:1:4, and being prepared as living bacteria count after stirring is 1.0X 109
The plastc ring of CFU/mL.
(3) regulation of filtrate viscosity
Concentration is the CaCl of 1% (w/v)2Aqueous solution, is 2000mPa s by the viscosity adjustment of filtrate, from
And obtain filemot complex micro organism fungicide.
(4) the one-tenth film viscosity of complex micro organism fungicide and the regulation of percentage elongation
Using Texture instrument, with reference to GB 16421-1996 standard, concentration is the Cacl of 1%2Aqueous solution and
The glycerol liquor liquid of 1%, by described microbial bacterial agent after 30 DEG C of thickness film forming 30 minutes with 0.5mm made
Viscosity and the percentage elongation of the film obtained are adjusted to 3Mpa and 1.5% (w/v) respectively, it is thus achieved that have biotic resistance and become
The complex micro organism fungicide of film.
Estimating of viscosity formula is as follows: TF=F/S
Wherein, TF is viscosity (g/cm2)；F is the maximum tension (g) that testing sample bears when rupturing；S
For the cross-sectional area (cm of testing sample before inspection2)
The computing formula of percentage elongation is as follows: and E%=(L-L ') × 100%L
Wherein, E is elongation at break (%)；L-L ' is the development length (mm) during film fracture.
The important technological parameters of biotic resistance film product is as follows:
Outward appearance: brown solution；
Solution viscosity is 2500mPa s；
Viscosity is 2Mpa to 3.6Mpa；
Percentage elongation is 1.2%；
Film-forming temperature is 30 DEG C.
Embodiment 2 utilizes the composite microbial bacteria with biotic resistance and film property obtained in embodiment 1
Agent sprays Fructus Mali pumilae
Bear fruit at Fructus Mali pumilae and start phase i.e. June 15, will there is the composite microbial bacteria of biotic resistance and film property
Agent is directly sprayed on fruit surface with the amount of spraying of 20Kg/ mu with aerosol apparatus, hereafter sprays once every 15 days,
Continuous 5 times.The most by the end of October, statistical measurement diseased fruit rate, single fruit weight, single plant yield, and respectively from
Each process five positions in plant East, West, South, North and win 5 fruits at random, survey its acidity, Vc content,
The indices such as hardness, are evaluated with regard to fruit quality.Test the most in triplicate, compares and protects into conventional bagging
Protecting, sack is buied from Luochuan fruit tree research, matched group bagging time with spray time consistency first.Fruit
Quality survey result is as shown in table 1 and Fig. 1.
Table 1 fruit quality survey result
Result shows, sprayed the fruit diseases of experimental group of the complex micro organism fungicide of the present invention a situation arises,
The key technical indexes such as single fruit weight, single plant yield is superior to bagging comparison fruit tree.Apple Bags technology is due to fruit
Tangible trophophase is constantly in no light condition and directly results in fruit solid content and the reduction of pol growing amount, and makes
Grow fruit tree single plant yield under field conditions (factors) with the fruit tree of experimental group and increase by 10.93%；Fruit pol increases
32.6%, solid content increase by 17.43%, do not detect any pesticide residues, the quality of fruit, mouthfeel, hardness,
The key technical indexes such as storage life are all significantly better than bagging fruit.
Embodiment 3: utilize the complex microorganism with biotic resistance and film property obtained in embodiment 1
Microbial inoculum sprays Fructus actinidiae chinensis
Main Pathogenic Bacteria (kiwi berry bacterial leaf blight pathogen and Fructus actinidiae chinensis antibacterial with the Kiwi growth phase
Property Pathogenic Fungus of Canker) be indicator bacteria, use flat board face-off method inspection biocontrol microorganisms bacillus subtilis (Bacillus
And the fungistatic effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) substils).Result shows
This two strains bacterium can effectively prevent and treat.
Using of complex micro organism fungicide.Bear fruit the phase at Fructus actinidiae chinensis, by produce complex micro organism fungicide with
The spray of 20Kg/ mu is directly sprayed on fruit surface in right amount with aerosol apparatus.Matched group is that conventional bagging processes.
Field control is tested.The biomembrane field control result of 1 year by a definite date shows, uses composite microbial bacteria
The growth of experimental group fruit tree tree vigo(u)r that agent sprays, shoot length and BAIYE fresh weight, a situation arises for disease, fruit
The key technical indexes such as size is homogeneous, commodity fruit rate are superior to bagging comparison fruit tree.Traditional bagging method by
It is constantly in no light condition in fruit at trophophase and directly results in fruit solid content and the reduction of pol growing amount,
And use biomembranous fruit tree to grow its chlorophyll under field conditions (factors) and increase 15%-25%, yield increases
20%-35%.Fruit pol increases 6%-12% after testing, and solid content increases 4%-108%, VC and increases by 40%,
Not detecting any pesticide residues, the key technical indexes such as the quality of fruit, mouthfeel, hardness, storage life is the biggest
It is better than greatly bagging fruit.
Claims (10)
1. the method preparing the complex micro organism fungicide with biotic resistance and film property, it is characterised in that
Described method includes:
(1) bacillus subtilis (Bacillus substils), bacillus amyloliquefaciens (Bacillus are cultivated respectively
Amyloliquefaciens) and leaf bacillus (Phyllobacterium sp), it is thus achieved that bacillus subtilis bacteria suspension, solution
Bacillus amyloliquefaciens suspension and leaf bacillus suspension；With
(2) by the mixing of described bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension all
Even, thus obtain plastc ring；
(3) it is more than or equal to 2000mPa s by the viscosity adjustment of described plastc ring, thus obtains
Described complex micro organism fungicide.
Method the most according to claim 1, it is characterised in that in step (1), by described withered
Grass bacillus cereus, described bacillus amyloliquefaciens and described leaf bacillus enter the most successively the cultivation temperature of 35 DEG C
The row Tube propagation of 72 hours, the shake-flask culture of 72 hours, the seed tank culture of 72 hours and 72 hours
Fermentor cultivation, thus respectively obtain described bacillus subtilis bacteria suspension, described bacillus amyloliquefaciens suspension
With described leaf bacillus suspension.
Method the most according to claim 1, it is characterised in that described bacillus subtilis bacteria suspension, institute
The living bacteria count stating bacillus amyloliquefaciens suspension and described leaf bacillus suspension is more than or equal to 109
CFU/mL。
Method the most according to claim 1, it is characterised in that described complex micro organism fungicide effective
Viable count is more than or equal to 109CFU/mL。
Method the most according to claim 1, it is characterised in that in step (2), described hay
The volume ratio of bacillus cereus suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension is 1:1:4.
Method the most according to claim 1, it is characterised in that described method also includes:
To described bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus before step (2)
Suspension carries out the step of screenings separation respectively；And/or
After step (2) and before step (3), described plastc ring is carried out screenings separation
Step.
7. the complex micro organism fungicide prepared by the method according to any one of claim 1 to 6.
Complex micro organism fungicide the most according to claim 7, it is characterised in that described microbial inoculum is at 30 DEG C
There is the viscosity of 2Mpa to 3.6Mpa with film obtained after the thickness film forming 30 minutes of 0.5mm and be more than
Or the percentage elongation equal to 1.2%.
9. the application in fruit is protected of the complex micro organism fungicide described in claim 7 or 8.
Application the most according to claim 9, it is characterised in that described complex micro organism fungicide is present
Really the phase sprays in fruit surface with the amount of 20,000 grams/acre.
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| CN114916358A (en) * | 2022-06-13 | 2022-08-19 | 山西工程科技职业大学 | Application of edible corn starch film in fruit bag |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114916358A (en) * | 2022-06-13 | 2022-08-19 | 山西工程科技职业大学 | Application of edible corn starch film in fruit bag |
| CN114916358B (en) * | 2022-06-13 | 2023-11-14 | 山西工程科技职业大学 | Application of edible corn starch film in fruit bagging |
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