CN105886435B - A kind of complex micro organism fungicide with biotic resistance and film forming and preparation method thereof and application - Google Patents
A kind of complex micro organism fungicide with biotic resistance and film forming and preparation method thereof and application Download PDFInfo
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- CN105886435B CN105886435B CN201610305515.1A CN201610305515A CN105886435B CN 105886435 B CN105886435 B CN 105886435B CN 201610305515 A CN201610305515 A CN 201610305515A CN 105886435 B CN105886435 B CN 105886435B
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Abstract
The present invention provides a kind of methods for preparing the complex micro organism fungicide with biotic resistance and film forming, the described method includes: (1) cultivates bacillus subtilis (Bacillus substils), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and leaf bacillus (Phyllobacterium sp) respectively, respective suspension is obtained；(2) each bacteria suspension is uniformly mixed, to obtain plastc ring；It (3) is more than or equal to 2000mPas, to obtain the complex micro organism fungicide by the viscosity-adjusting agent of the plastc ring.The present invention also provides the application of the complex micro organism fungicide as made from the method and the complex micro organism fungicide in fruit protection.The present invention solves the problems such as wasting of resources caused by bagging and environmental pollution during Production of fruit, simultaneously, the usage amount of pesticide is greatly reduced in its significant biocontrol effect, provides novel, environmentally friendly, safe and efficient fruit industry production mode to develop green agriculture.
Description
Technical field
The present invention relates to belong to field of biological control, and in particular to a kind of composite microbial with biotic resistance and film forming
Object microbial inoculum and preparation method thereof and the application in fruit protection.
Background technique
In modern fruit industry, the crop failure due to caused by disease infection and chemical pesticide bring environment is excessively used
The problems such as deterioration and food safety crisis is the significant obstacle for endangering human health, restricting agricultural sustainable development.Wherein, to water
The prevention and treatment of fruit disease evil is key problem.
Currently, domestic mostly using spraying chemical pesticide and to technical methods such as fruit baggings come controlling disease.But these
There are still following points for method: although the infection of germ can be effectively suppressed in chemical pesticide, but long-term, excessive use not only can be right
Environment pollutes, and the remaining pesticide of crop surface can also threaten food safety and damage human health.In addition, being used for a long time also
It is easy to cause fungi to develop drug resistance and brings many new problems to agricultural production.To reduce direct pollution of the pesticide to fruit
And protect fruit from the damage of extraneous various factors, the bagging technique based on physical control is made extensively in current apple production
Method.But there is also many disadvantages for this method: firstly, bagging, which will lead to fruit, can not receive illumination in growth period, making sugar
Divide equal organic matters synthetic quantity to reduce, and then influence the mouthfeel and quality of fruit, reduces its commodity value.Secondly as current
Bagging goes the operation of bag to mostly use artificial progress, this had both increased artificial demand, and had also increased production cost.Further, since
Bagging can not Reusability, burning disposal is made after, causes the huge wasting of resources and environmental pollution.Therefore, one kind is found
Safely, effectively with economical and practical method, make it that can effectively prevent disease, ensures that fruit safety, improves fruit quality
And reduce production cost, it has also become urgent problem to be solved in green high-efficient fruit industry process of sustainable development.
Summary of the invention
In order to overcome the problems referred above, the object of the present invention is to provide a kind of complex microorganism with biotic resistance and film forming
Microbial inoculum and preparation method thereof and the application in fruit protection.The complex micro organism fungicide is sprayed on fruit, can be replaced
The method of generation traditional bagging, can effectively prevent disease, ensure that fruit safety, improve fruit quality.
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 be with
Efficiently inhibit various fungal diseases biocontrol microorganisms and can extracellular polysaccharide production sugar bacterium.The present inventor is by obtained biocontrol microorganisms and production
Sugared bacterium carries out scientific compounding and complex micro organism fungicide simultaneously is made, so as to effectively play their synergistic effect.It is described compound
Microbial bacterial agent is metabolizable during the fermentation to generate various bioactive substances.These substances are effectively inhibiting infecting for germ
While with enhancing crop resistance, also there is good film forming, gas permeability and ductility, being sprayed on fruit surface can be fast
Speed forms one layer of thin semi-permeable membrane in fruit face, can fix complex microorganism antagonism bacterium solution, and fruit surface can be prevented impaired；Together
When, gas permeability makes fruit that can normally receive illumination and breathing, on fruit growth and quality without influence.Therefore, using this hair
It is bright to can reach safe and effective prevention target.
The present invention provides in first aspect and a kind of prepares complex micro organism fungicide with biotic resistance and film forming
Method, which comprises (1) cultivate bacillus subtilis (Bacillus substils), bacillus amyloliquefaciens respectively
It is outstanding to obtain bacillus subtilis for (Bacillus amyloliquefaciens) and leaf bacillus (Phyllobacterium sp)
Liquid, bacillus amyloliquefaciens suspension and leaf bacillus suspension；(2) by the bacillus subtilis bacteria suspension, bacillus amyloliquefaciens
Suspension and leaf bacillus suspension are uniformly mixed, to obtain plastc ring；
(3) by the viscosity-adjusting agent of the plastc ring be more than or equal to 2000mPas, such as 2000 to
3000mPas, to obtain the complex micro organism fungicide.
The present invention provides complex micro organism fungicide made from the method as described in first aspect present invention in second aspect.
The present invention provides complex micro organism fungicide described in second aspect of the present invention in fruit protection in the third aspect
Application.
The present invention has the advantage that
1, control efficiency is good, security performance is high
Complex micro organism fungicide provided by the invention has biotic resistance and film forming, and has safe and nontoxic spy
It is fixed, it both can guarantee control efficiency in application fruit protection, ensure that the safety of fruit.
2, fruit quality is good, yield is high
Complex micro organism fungicide of the present invention has output of sugar is high, produces short sugared period, good film-forming property and performance to stablize
The features such as and advantage, and there is fine air permeability and ductility after forming a film, not only can rapid film formation with effectively fixed resistance object
Matter, and protective layer can be formed to prevent fruit surface impaired in fruit surface, meanwhile, gas permeability normally receive fruit can
Illumination and breathing, help to improve crop yield and quality.
3, safe and non-toxic pollution-free, sustainable development
It is nontoxic edible that complex micro organism fungicide of the invention is formed by biotic resistance membrane safety, will not both pollute ring
Border will not damage human health.The use of the invention can effectively protect ecological environment, ensures food safety,.
4, production cost is reduced
Complex micro organism fungicide of the present invention is easy to field operation, demand pole of the whole operation process to manpower
It is small, thus production cost is greatly lowered.
5, facilitate the foundation of the ecological agriculture
The performance of the biomembrane formed by complex micro organism fungicide of the present invention is extremely stable, is used for fruit
Biological control can both effectively prevent disease, ensure that the quality and yield of crop.Promotion and application of the invention help to build
Safe and environment-friendly, green, efficient completely new fruit industry production mode is found, and the sustainable development of ecological fruit industry can be pushed.
Detailed description of the invention
Fig. 1 fruit tree growth situation (left side is experimental group, and right side is bagging control group)；With
Fig. 2 fruit is apparent (left side is experimental group, and right side is bagging control group).
Specific embodiment
As described above, the present invention first aspect provide it is a kind of prepare it is compound micro- with biotic resistance and film forming
The method of bacteria agent, which comprises
(1) bacillus subtilis (Bacillus substils), bacillus amyloliquefaciens (Bacillus are cultivated respectively
Amyloliquefaciens) and leaf bacillus (Phyllobacterium sp), acquisition bacillus subtilis bacteria suspension solve starch bud
Spore bacillus suspension and leaf bacillus suspension；
(2) the bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension are uniformly mixed, thus
Obtain plastc ring；
It (3) is more than or equal to 2000mPas, to obtain described multiple by the viscosity-adjusting agent of the plastc ring
Close microbial bacterial agent.
Bacillus subtilis is the type culture of bacillus, its metabolizable generation protease, lipase, amylase etc.
Multiple active enzymes are used for the preparation of animal feed, can effectively facilitate the degradation of nutrient in feed, improve its nutrient absorption
Rate；In addition, it can produce the antibacterial materials such as iturin, Surfactin, polymyxins during the growth process, by it
Disease control for crop can be effectively suppressed the activity of all kinds of bacterium opportunistic pathogens and enhance the inducible resistance of system of crop.
Bacillus amyloliquefaciens are a kind of same bacillus subtilis affiliation and its close bacterial strain, it is distributed wide, easy
Culture, resistance is strong, breeding is fast and can generate a large amount of metabolites in growth course, the latter there is wide spectrum to bacterium, fungi and
Efficient inhibiting effect.Therefore, bacillus amyloliquefaciens have become current biological as the biocontrol bacterial strain of great application potential and prevent
Control the research hotspot of plant disease.
Leaf bacillus is a kind of aerobic Gram-negative bacteria, and the metabolizable various polysaccharide of generation form sugared quilt, is used for resisting
The preparation of property film can rapid film formation；In addition, the bacterium does not hydrolyze starch, pectin and cellulose, therefore the use of the bacterium will not destroy fruit
Real quality.
It is a discovery of the invention that these three bacterium are prepared into complex microbial inoculum after science compounding.Gained microbial inoculum, which has, to be produced
Sugar amount is high, produces the features such as short sugared period, good film-forming property and performance are stablized and advantage, and has fine air permeability after forming a film and prolong
Malleability, not only can rapid film formation with effectively fixed resisting substance, and protective layer can be formed in fruit surface to prevent fruit surface
It is impaired.
Strain used herein is known strain, can be obtained by conventional screening, commercial means or other approach.
In one embodiment, it can be obtained through commercial channels strain as described herein, such as bacillus subtilis can
It, selectively, can be from Xi'an Zi Rui Biotechnology Co., Ltd (to be bacillus subtilis (Bacillus subtilis)
Location: postcode: 722300) Shaanxi Province, riverfront new district, the Mei County main road Mei Wu eastern section obtains above-mentioned bacterial strains.
In one embodiment, strain as described herein, such as bacillus amyloliquefaciens can be obtained through commercial channels
It can be bacillus amyloliquefaciens (Bacillus amyloliquefaciens), it selectively, can be from the purple auspicious biological section in Xi'an
(address: postcode: 722300) Shaanxi Province, riverfront new district, the Mei County main road Mei Wu eastern section obtains above-mentioned bacterial strains for skill Co., Ltd.
In one embodiment, it can be obtained through commercial channels strain as described herein, such as leaf bacillus can be leaf
Bacillus (Phyllobacterium sp) selectively can be from Xi'an Zi Rui Biotechnology Co., Ltd (address: Shaanxi Province's eyebrow
Postcode: 722300) county, new district, the riverfront main road Mei Wu eastern section obtains above-mentioned bacterial strains.
In some embodiments, in step (1), by the bacillus subtilis, the bacillus amyloliquefaciens and
The leaf bacillus successively carries out 72 hours Tube propagations, shaking flask culture in 72 hours, 72 small in 35 DEG C of cultivation temperature respectively
When seed tank culture and 72 hours fermentation tank cultures, to respectively obtain the bacillus subtilis bacteria suspension, the Xie Dian
Afnyloliquefaciens suspension and the leaf bacillus suspension.
In some embodiments, the bacillus subtilis bacteria suspension, the bacillus amyloliquefaciens suspension and the leaf
The living bacteria count of bacillus suspension is more than or equal to 109CFU/mL is greater than or is equal to 1X109CFU/mL、
5X109CFU/mL、1X1010CFU/mL or 5X109CFU/mL。
In some embodiments, the living bacteria count of the complex micro organism fungicide is more than or equal to 109CFU/
ML is greater than or is equal to 1X109CFU/mL、5X109CFU/mL、1X1010CFU/mL or 5X109CFU/mL。
In some embodiments, in step (2), the bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension
Volume ratio with leaf bacillus suspension is 1:1:2~1:1:5, optimal proportion 1:1:4.
In some embodiments, viscosity is adjusted using calcium chloride solution and glycerite, in some preferred implementations
In mode, the calcium chloride solution (w/v) and/or the glycerite for being 1% (w/v) for being 1%.
The present invention provides complex micro organism fungicide made from the method as described in first aspect present invention in second aspect.
In some embodiments, microbial inoculum film obtained after 30 DEG C of thickness with 0.5mm forms a film 30 minutes has 2Mpa extremely
The viscosity of 3.6Mpa (for example, 2,2.5,3,3.6MPa) and the elongation more than or equal to 1.2% (are greater than or wait
In 1.2,1.4,1.6,1.8 or 2.0 elongation).
The present invention provides the complex micro organism fungicide or this hair made from first aspect present invention in the third aspect
Application of the complex micro organism fungicide described in bright second aspect in fruit protection.
The present invention is not particularly limited the method for application of the complex micro organism fungicide, but in some preferred realities
It applies in mode, is applied by the way of spraying.In some preferred embodiments, the complex micro organism fungicide is in fruit setting
Phase is sprayed on fruit surface with 20,000 grams/acre of amount.In some embodiments, the fruit is selected from by apple and Kiwi berry group
At group.
Embodiment
It is specific implementation example of the invention below, with invention is further described in detail, but implementation of the invention
Mode is without being limited thereto.
The bacillus subtilis (Bacillus substils) that is in embodiment, bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens it) is bought with leaf bacillus (Phyllobacterium sp) from Xi'an Zi Rui Biotechnology Co., Ltd
It obtains.Used medium be beef extract-peptone fluid nutrient medium (5g beef extract, 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 successively cultivated as follows in 35 DEG C of difference:
Tube propagation-shaking flask culture-fermentation tank culture, used medium are beef extract-peptone fluid nutrient medium (5g ox
Meat extract, 10g peptone, 5g NaCl, 15g agar, 1000mL water), incubation time is 72h, finally respectively obtains effective viable bacteria
Number is 109Bacillus subtilis bacteria suspension, bacillus amyloliquefaciens bacteria suspension and the leaf bacillus bacteria suspension of CFU/mL or more.
(2) preparation of plastc ring
By the resulting bacillus subtilis bacteria suspension of fermentation tank culture, bacillus amyloliquefaciens bacteria suspension and leaf bacillus
Bacteria suspension is mixed with the volume ratio of 1:1:4, and being prepared into living bacteria count after mixing evenly is 1.0X 109The Mixed Microbes of CFU/mL
Suspension.
(3) adjusting of filtrate viscosity
The CaCl for the use of concentration being 1% (w/v)2The viscosity-adjusting agent of filtrate is 2000mPas, to obtain by aqueous solution
Filemot complex micro organism fungicide.
(4) adjusting at film viscosity and elongation of complex micro organism fungicide
Using Texture instrument, referring to GB 16421-1996 standard, the Cacl for the use of concentration being 1%2Aqueous solution and 1% it is sweet
Oil-water fluid, by the viscosity of microbial bacterial agent film obtained after 30 DEG C of thickness with 0.5mm forms a film 30 minutes and elongation
Rate is adjusted to 3Mpa and 1.5% (w/v) respectively, obtains the complex micro organism fungicide with biotic resistance and film forming.
The calculation formula of viscosity is as follows: TF=F/S
Wherein, TF is viscosity (g/cm2)；F is the maximum tension (g) born when sample to be tested ruptures；S is to be measured before examining
Cross-sectional area (the cm of sample2)
The calculation formula of elongation is as follows: E%=(L-L ') × 100%L
Wherein, E is elongation at break (%)；Development length (mm) when L-L ' is film fracture.
The important technological parameters of biotic resistance film product are as follows:
Appearance: brown solution；
Solution viscosity is 2500mPas；
Viscosity is 2Mpa to 3.6Mpa；
Elongation is 1.2%；
Film-forming temperature is 30 DEG C.
Embodiment 2 is sprayed using the complex micro organism fungicide with biotic resistance and film forming obtained in embodiment 1
Apple
Bear fruit in apple and start phase i.e. June 15, by the complex micro organism fungicide with biotic resistance and film forming with
20Kg/ mus of the amount of spraying directly is sprayed on fruit surface with sprayer, hereafter sprays every 15 days once, continuous 5 times.In current year
By the end of October, statistical measurement diseased fruit rate, single fruit weight, single plant yield, and five sides from each processing plant East, West, South, North respectively
5 fruits are won at random in position, survey the indices such as its acidity, Vc content, hardness, are evaluated with regard to fruit quality.Test counterpoise
Multiple to compare three times as conventional protected by bagging, sack is bought from Luochuan fruit tree research, control group bagging time with when spraying for the first time
Between it is consistent.Fruit quality investigation result is as shown in table 1 and Fig. 1.
1 fruit quality investigation result of table
The result shows that having sprayed the fruit diseases of the experimental group of complex micro organism fungicide of the invention, a situation arises, single fruit
The key technical indexes such as weight, single plant yield are superior to bagging control fruit tree.Apple Bags technology due to fruit growth period always
Directly resulting in fruit solid content and pol production quantity in no light condition reduces, and uses the fruit tree of experimental group in natural conditions
Lower growth fruit tree single plant yield increases by 10.93%；Fruit pol increases by 32.6%, and solid content increases by 17.43%, is not detected any
Pesticide residue, the key technical indexes such as quality, mouthfeel, hardness, the storage life of fruit are significantly better than bagged fruit.
Embodiment 3: it is sprayed using the complex micro organism fungicide with biotic resistance and film forming obtained in embodiment 1
Apply Kiwi berry
With main pathogenic bacteria (kiwi berry bacterial leaf blight pathogen and the kiwi berry bacterial ulcer of Kiwi growth phase
Sick pathogen) it is indicator bacteria, biocontrol microorganisms bacillus subtilis (Bacillus substils) reconciliation is examined using tablet face-off method
The fungistatic effect of bacillus amyloliquefaciens (Bacillus amyloliquefaciens).This two plants of bacterium can effectively prevent as the result is shown
It controls.
The application of complex micro organism fungicide.Bear fruit the phase in Kiwi berry, by the complex micro organism fungicide of production with 20Kg/ mus
Spray is directly sprayed on fruit surface with sprayer in right amount.Control group is conventional bagging processing.
Field control experiment.Biomembrane field control in 1 year by a definite date using complex micro organism fungicide the results show that sprayed
The growth of experimental group fruit tree tree vigo(u)r, a situation arises, fruit size is uniform, commodity fruit rate for shoot length and blinds fresh weight, disease
Etc. the key technical indexes be superior to bagging control fruit tree.Traditional bagging method in growth period is constantly in no light due to fruit
State directly results in fruit solid content and pol production quantity and reduces, and grows its leaf under field conditions (factors) using the fruit tree of biomembrane
Green element increases 15%-25%, and yield increases 20%-35%.Increase 6%-12% through detection fruit pol, solid content increases 4%-
108%, VC increase by 40%, and any pesticide residue, the key technical indexes such as quality, mouthfeel, hardness, the storage life of fruit are not detected
It is significantly better than bagged fruit.
Claims (13)
1. a kind of method for preparing the complex micro organism fungicide with biotic resistance and film forming, which is characterized in that the method
Include:
(1) bacillus subtilis (Bacillus substils), bacillus amyloliquefaciens (Bacillus are cultivated respectively
Amyloliquefaciens) and leaf bacillus (Phyllobacterium sp), acquisition bacillus subtilis bacteria suspension solve starch bud
Spore bacillus suspension and leaf bacillus suspension；With
(2) the bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension are uniformly mixed, to obtain
Plastc ring；
It (3) is more than or equal to 2000mPas, to obtain described compound micro- by the viscosity-adjusting agent of the plastc ring
Bacteria agent.
2. the method according to claim 1, wherein in step (1), by the bacillus subtilis, described
Bacillus amyloliquefaciens and the leaf bacillus 35 DEG C of cultivation temperature successively carry out respectively 72 hours Tube propagation, 72 hours
Shaking flask culture, 72 hours seed tank cultures and 72 hours fermentation tank cultures, to respectively obtain the bacillus subtilis
Bacteria suspension, the bacillus amyloliquefaciens suspension and the leaf bacillus suspension.
3. the method according to claim 1, wherein the bacillus subtilis bacteria suspension, the solution starch gemma
The living bacteria count of bacillus suspension and the leaf bacillus suspension is more than or equal to 109CFU/mL。
4. the method according to claim 1, wherein the living bacteria count of the complex micro organism fungicide be greater than
Or it is equal to 109CFU/mL。
5. the method according to claim 1, wherein in step (2), the bacillus subtilis bacteria suspension is solved
The volume ratio of bacillus amyloliquefaciens suspension and leaf bacillus suspension is 1:1:4.
6. the method according to claim 1, wherein the method also includes:
Before the step (2) to the bacillus subtilis bacteria suspension, bacillus amyloliquefaciens suspension and leaf bacillus suspension respectively into
The step of row screenings separates；And/or
After step (2) and the step of carrying out screenings separation to the plastc ring before step (3).
7. the method according to claim 1, wherein being adjusted using calcium chloride solution and glycerite described multiple
Close microbial bacterial agent at film viscosity and film forming elongation.
8. the method according to the description of claim 7 is characterized in that the calcium chloride solution be 1 weight % aqueous solution and/or
The glycerite is the aqueous solution of 1 volume %.
9. the complex micro organism fungicide as made from method described in any item of the claim 1 to 8.
10. complex micro organism fungicide according to claim 9, which is characterized in that the microbial inoculum is in 30 DEG C of thickness with 0.5mm
Degree film forming viscosity of the film with 2Mpa to 3.6Mpa obtained after 30 minutes and the elongation more than or equal to 1.2%.
11. application of the complex micro organism fungicide described in claim 9 or 10 in fruit protection.
12. application according to claim 11, which is characterized in that the complex micro organism fungicide is in setting phase with 20,000
Gram/acre amount be sprayed on fruit surface.
13. application according to claim 11 or 12, which is characterized in that the fruit is selected from and is made of apple and Kiwi berry
Group.
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| CN104496573A (en) * | 2014-12-04 | 2015-04-08 | 上海绿乐生物科技有限公司 | Special biocontrol compound microbial fertilizer for grapes and production method and application |
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- 2016-05-10 CN CN201610305515.1A patent/CN105886435B/en active Active
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| CN101418268A (en) * | 2007-10-26 | 2009-04-29 | 中国海洋大学 | Production of novel bacterial exopolysaccharides by using Phyllobacterium sp.nov.921F bacterial strain |
| CN104496573A (en) * | 2014-12-04 | 2015-04-08 | 上海绿乐生物科技有限公司 | Special biocontrol compound microbial fertilizer for grapes and production method and application |
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