CN106498022A - A kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi quickly lures anti-screening technique - Google Patents
A kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi quickly lures anti-screening technique Download PDFInfo
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Abstract
The invention discloses a kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape Antagonistic Fungi quickly lures anti-screening technique, the method is using the light grey and pinkish fungal hyphae body powder of inactivation, chitin, shitosan, Ca2+、Mg2+、Zn2+Culture medium is added Deng as bacterial strain antagonistic metabolite inducer;Using 3~4 days concentration 10 of culture4The grey mold spore liquid of cfu/mL is inverted against long flat board fast spraying botrytis cinerea spore liquid, after 1h and is put in 28 DEG C of constant temperature co-cultivation 48h 72h, observes its growing state;And the Antagonism of Antagonistic Fungi is determined by filter paper enzyme secondary screening.What the present invention provided a kind of high biological and ecological methods to prevent plant disease, pests, and erosion power botrytis cinerea Antagonistic Fungi of grape quickly lures anti-screening technique, it is suitable to the screening of the fruits and vegetables botrytis cinerea efficient stable Antagonistic Fungi such as grape, operation is simple compared with conventional method, quick, screening efficiency is high, biological control for botrytis cinerea after grape harvest lays the foundation, there is provided reliable technology and theoretical foundation.
Description
Technical field
The invention belongs to the fresh-keeping technical field of biological control of postharvest fruit and vegetable, more particularly to a kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea
Antagonistic Fungi quickly lures anti-screening technique.
Background technology
Botrytis cinerea (Botrytis cinerea), also known as the pathogen of Botrytis cinerea, is a kind of wide host's property, can cause 200
The downright bad auxotype disease fungus of multiple known plants (such as water fruits and vegetables and flowers) gray mold.Which is widely distributed in atmosphere,
Field crops can not only be infected, equally can be brought about great losses to the rear stage of adopting of plant.The fruits and vegetables run in daily life
Put and can become mildewed for a period of time, majority may receive infecting for botrytis cinerea.Cut-off 2013, in the world it is not yet found that appointing
A kind of what plant produces resistance to botrytis cinerea.
Botrytis cinerea fructification is born from mycelia or sclerotium;Conidiophore 280~550*12~24 micron, grow thickly, ash
Color, after switch to brown, its ultimate swelling or taper have little projection thereon;Conidium is singly born on kick;Point
Spherical or avette, 9~15*6.5~10 micron in raw spore Asia.
Botrytis cinerea can (0 DEG C) growth under cryogenic, propagated by producing substantial amounts of grey conidium, with which
Its postharvest pathogenic fungi is compared, with the advantage that latent infection and low temperature cause a disease.Meanwhile, which also has breeds fast, hereditary variation
The characteristics of big and grade of fit is high.
At present, the pollution of the postharvest fruit and vegetable such as grape, Chinese cabbage botrytis cinerea is the principal element for affecting its preservation quality and safety.Often
Using cylinder-plate method secondary screening being used again after the mixed bacterium spread plate primary dcreening operation of test tube more than antagonism bacterial screening method, move block method sieve with agar
Choosing, filter paper enzyme, opposite culture method etc..Time-consuming for these methods, when Antagonistic Fungi is co-cultured with pathogen, sometimes due to cause of disease
The growth of bacterium comparatively fast covered Antagonistic Fungi, it is also possible to when Antagonistic Fungi does not discharge mortifier, just by cause of disease cap mistake.
Therefore, botrytis cinerea biological control method is developed, and particularly Antagonistic Fungi rapid screening method is imperative.
According to retrieval, Antagonistic Fungi rapid screening method related to the application as follows is found, specifically disclosed content is as follows:
Patent document CN103409494A discloses a kind of rapidly and efficiently screening technique of fusarium antagonistic bacteria, using flat board two
Step cultivation carries out the screening of Antagonistic Fungi, and then separation of bacterial first on nutrient medium is sprayed on the germy flat board of length
The spore liquid of mist sickle-like bacteria, cultivates under the conditions of continuing at 28 DEG C, and the inhibition zone that observes around bacterium such as occurs antibacterial around
Circle, illustrates that the bacterium has antagonistic ability to sickle-like bacteria.The present invention adopts same flat board two-steps tissue culture method, to separating from sample
Bacterium out need not carry out proceeding to another medium culture measure one by one after purification again on nutrient medium, greatly save
The culture medium that wants needed for experimentation, saves manpower, material resources and financial resources, improves the screening efficiency of Antagonistic Fungi.
Patent document CN104328059A discloses the screening technique that a kind of mulberry fruit surface causes rotten Antagonistic Fungi, including following step
Suddenly:Step 1, the preparation of antagonism bacterium solution:The intact mulberry fruit of cleaning is taken, is placed in after chopping in SPSS, mixed liquor is permanent
Warm shaken cultivation, gained nutrient solution are antagonism bacterium solution;Step 2, antagonism bacterium colony group are cultivated:Step 1 gained antagonism bacterium solution is used
SPSS dilution obtains dilution, takes 1mL dilutions and coats in PDA culture medium, incubated, obtains bacterium colony group;
Step 3, single bacterium colony culture:Picking step 2 gained bacterium colony group, being seeded in PDA culture medium carries out pure culture, and repeatedly separately 3~5
Secondary, you can to obtain Antagonistic Fungi single bacterium colony.
Patent document CN104694407A discloses a kind of screening technique of melon fruit surface Antagonistic Fungi, and the present invention is right first
The separation of fruit surface bacterium, then carries out the screening of Antagonistic Fungi, finally carries out after antibacterial Gram's staining in electron microscope
Lower observed and recorded Antagonistic Fungi filters out the best antagonism of fungistatic effect to alternaric bacteria on muskmelon and the antagonism of sickle-like bacteria
Bacterium.
There is larger difference with the application in above-mentioned screening technique, do not affect the novelty and creativeness of the application.
Content of the invention
It is an object of the invention in place of overcoming the deficiencies in the prior art, there is provided a kind of simple structure, fast and efficiently high
Biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi quickly lures anti-screening technique.
The present invention realizes that the technical scheme of purpose is as follows:
A kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi quickly lures anti-screening technique, and step is as follows:
(1) dilution variable concentrations, the different dilution dilutions after concussion mixing in sterilized water by the grape for having Mechanical wound
Liquid is respectively coated in the light grey and pinkish fungal hyphae body powder, 0.5-lwt% chitins, 0.5-lwt% inactivated containing 2-5wt%
Shitosan and the Ca of 0.05-0.1wt%2+、Mg2+、Zn2+On the PDA culture medium flat board of salt, each dilution is repeated twice, and will apply
The good culture dish of cloth is inverted under the conditions of being put in 28 DEG C and cultivates 48h, and after selecting coating culture, length has the flat board of Antagonistic Fungi standby;
By step (1) in the remaining botrytis cinerea PDA plate without picking in culture 5-7d to a large amount of spores generation after,
Lower botrytis cinerea Spores are washed with 0.5wt% Tween-80s, grey mold spore liquid is obtained, blood counting chamber adjusts spore liquid miospore
Concentration be 104cfu/mL;
(3) the ratio according to 0.1-0.3wt% in the grey mold spore liquid that concentration is regulated adds N- acetyl glucosamines and several
Fourth oligosaccharides, against step (1) in the length selected have Antagonistic Fungi flat board fast spraying once, spray concentration is 104Cfu/mL grey mold
Bacterium spore liquid, is inverted after 1h and is put in 28 DEG C of incubated 48h-72h, and the inhibition zone size after observation co-cultivation, according to inhibition zone
Size screening Antagonistic Fungi, inhibition zone size be inhibition zone external diameter subtract button go cultivate Antagonistic Fungi radius, the bigger theory of inhibition zone
Bright antagonistic effect is better;
(4) the bacterium with inhibition zone is carried out line purifying on nutrient medium flat board, until the bacterium colony on each flat board
Assume identical color, size and shape, the Antagonistic Fungi for as purifying;
(5) secondary screening:By the Antagonistic Fungi dibbling for having purified on nutrient medium flat board, in 28 DEG C of incubators after culture 48h,
Continue culture 48h after spray botrytis cinerea spore liquid in 28 DEG C of incubators, observation have stable inhibition zone for botrytis cinerea Antagonistic Fungi.
And, step nutrient medium (4) is wort agar culture medium or NYDA solid mediums;NYDA liquid
The formula of body culture medium is:Nutrient broth 8g, yeast extract 5.0g, glucose 10g, agar 18g, distilled water 1000mL, 121 DEG C go out
Bacterium 20min.
And, described nutrient medium is PDA liquid medium or PDA solid mediums;PDA liquid medium is matched somebody with somebody
Fang Wei:Potato 300g, glucose 20g, chloramphenicol 0.1g, distilled water 1000mL, add agar in PDA liquid medium
20g forms PDA solid mediums.
The advantage of the application and good effect are as follows:
What the application was provided quickly lures anti-screening technique when Antagonistic Fungi is screened, and adds 2%- in initial culture medium
The light grey and pinkish fungal hyphae body powder of 5% inactivation, 0.5%-l% chitins, the shitosan of 0.5%-l%, above-mentioned three kinds of materials
After organic cooperation, can assist in Antagonistic Fungi and grown, instead of original culture medium, the Antagonistic Fungi speed of growth is slow,
Lead to not the drawbacks of screening, effectively increase the screening efficiency of Antagonistic Fungi.
The application provide quickly lure anti-screening technique in the secondary screening of Antagonistic Fungi culture bacterium also in add N- acetyl
Glucose and chitin oligo saccharide, this in two material can increase the speed of growth of Antagonistic Fungi, be found through experiments, in the same time
In, than adding during single substance, inhibition zone wants big 25%, makes the selection result more directly perceived, improves screening efficiency.
This method is using the light grey and pinkish fungal hyphae body powder, chitin, shitosan, Ca for inactivating2+、Mg2+、Zn2+Deng as
Bacterial strain antagonistic metabolite inducer adds culture medium, makes the bacterial strain active ingredient with antagonism fully efficient in the medium
Release metabolite;After culture 3~4 days, using concentration 104The grey mold spore liquid of cfu/mL is against long flat board fast spraying grey mold
Bacterium spore liquid, is inverted after 1h and is put in 28 DEG C of constant temperature co-cultivation 48h-72h, observe its growing state, quick according to inhibition zone size
Primary dcreening operation grape botrytis cinerea Antagonistic Fungi;And the Antagonism of Antagonistic Fungi is determined through secondary screening.
What the present invention provided a kind of high biological and ecological methods to prevent plant disease, pests, and erosion power botrytis cinerea Antagonistic Fungi of grape quickly lures anti-screening technique, is suitable to the fruit such as grape
The screening of vegetable botrytis cinerea efficient stable Antagonistic Fungi, operates, quick, screening efficiency simple compared with conventional method high, is ash after grape harvest
The biological control of mould lays the foundation, there is provided reliable technology and theoretical foundation.
Specific embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
Following percentage is weight percentage, without specified otherwise thus, except having specified otherwise.
The method is using the light grey and pinkish fungal hyphae body powder, chitin, shitosan, Ca for inactivating2+、Mg2+、Zn2+Deng as
Bacterial strain antagonistic metabolite inducer adds culture medium;Using 3~4 days concentration 10 of culture4The grey mold spore liquid of cfu/mL is against length
Flat board fast spraying botrytis cinerea spore liquid, is inverted after 1h and is put in 28 DEG C of constant temperature co-cultivation 48h-72h, observe its growing state, root
According to photographic fog bacteria growing and the quick primary dcreening operation grape botrytis cinerea Antagonistic Fungi of inhibition zone size;And Antagonistic Fungi is determined by filter paper enzyme secondary screening
Antagonism.What the present invention provided a kind of high biological and ecological methods to prevent plant disease, pests, and erosion power botrytis cinerea Antagonistic Fungi of grape quickly lures anti-screening technique, is suitable to grape etc.
The screening of fruits and vegetables botrytis cinerea efficient stable Antagonistic Fungi, operates, quick, screening efficiency simple compared with conventional method high, after being grape harvest
The biological control of botrytis cinerea lays the foundation, there is provided reliable technology and theoretical foundation.
A kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi quickly lures anti-screening technique, and step is as follows:
(1) the grape for having Mechanical wound is configured to 10 after concussion mixing in sterilized waterOne 3, 10One 5, 10One 7Dilution,
Different dilution dilutions are respectively coated several in the light grey and pinkish fungal hyphae body powder, 0.5%-l% inactivated containing 2%-5%
The Ca of the shitosan and 0.05%-0.1% of Ding Zhi, 0.5%-l%2+、Mg2+、Zn2+On the PDA culture medium flat board of salt etc., each
Dilution is repeated twice, and coated culture dish is inverted under the conditions of being put in 28 DEG C and cultivates 48h, and after selecting coating culture, length has short of money
The flat board of antibacterial is standby (having Antagonistic Fungi circle on flat board);
By step (1) in the remaining botrytis cinerea PDA plate without picking in culture 5-7d to a large amount of spores generation after,
Lower botrytis cinerea Spores are washed with 0.5% Tween-80, grey mold spore liquid is obtained, blood counting chamber adjusts spore liquid miospore
Concentration is 104cfu/mL;
(3) the ratio according to 0.1-0.3wt% in the grey mold spore liquid that concentration is regulated adds N- acetyl glucosamines and several
Fourth oligosaccharides, against step (1) in the length selected have Antagonistic Fungi flat board fast spraying once, spray concentration is 104Cfu/mL grey mold
Bacterium spore liquid, is inverted after 1h and is put in 28 DEG C of incubated 48h-72h, and the inhibition zone size after observation co-cultivation, according to inhibition zone
Size screening Antagonistic Fungi, inhibition zone size be inhibition zone external diameter subtract button go cultivate Antagonistic Fungi the radius (radius of Antagonistic Fungi
For the size containing inhibition zone in picking rear plate in step), the bigger explanation antagonistic effect of inhibition zone is better;
(4) the bacterium with inhibition zone, in nutrient medium, (nutrient medium is wort agar culture medium or NYDA solids
Culture medium) line purifying is carried out on flat board, until the bacterium colony on each flat board assumes identical color, size and shape, as
The Antagonistic Fungi for having purified;
(5) secondary screening:By the Antagonistic Fungi dibbling for having purified on nutrient medium flat board, in 28 DEG C of incubators after culture 48h,
Continue culture 48h after spray botrytis cinerea spore liquid in 28 DEG C of incubators, observation have stable inhibition zone for botrytis cinerea Antagonistic Fungi, secondary screening
Inhibition zone parallel laboratory test error size is not more than 0.25cm, and equality result is stable;
Generally, inhibition zone >=2.0cm fungistatic effects are preferable.
Described nutrient medium is wort agar culture medium or NYDA solid mediums;NYDA fluid nutrient mediums are matched somebody with somebody
Fang Wei:Nutrient broth 8g, yeast extract 5.0g, glucose 10g, agar 18g, distilled water 1000mL, 121 DEG C of sterilizing 20min.
Step (1) and (2) in nutrient medium be PDA liquid medium or PDA solid mediums;PDA liquid medium
Formula be:Potato 300g, glucose 20g, chloramphenicol 0.1g, distilled water 1000mL, add fine jade in PDA liquid medium
Fat 20g forms PDA solid mediums.
Relevant experimental data explanation:
1st, in the application using 2%-5% inactivation light grey and pinkish fungal hyphae body powder, 0.5%-l% chitins,
For cultivating pathogenic bacteria and Antagonistic Fungi in the original PDA culture medium that the shitosan of 0.5%-l% is added, inhibition zone is turned out
Success rate improves 90-100%, iff PDA culture medium is used, it is difficult to turn out inhibition zone, and is difficult to observe.
2nd, in the application in the screening of Antagonistic Fungi, N- acetyl glucosamines and chitin oligo saccharide are added in the medium, can
The speed of growth of inhibition zone is improved, and 50-60% is improved than the speed of growth of blank, and inhibition zone wants big 10-25%.
Claims (3)
1. a kind of high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi quickly lures anti-screening technique, it is characterised in that:Step is as follows:
(1), by the grape for having Mechanical wound in variable concentrations are diluted after concussion mixing in sterilized water, different dilution dilutions divide
Do not coat the light grey and pinkish fungal hyphae body powder containing 2-5wt% inactivations, 0.5-lwt% chitins, the shell of 0.5-lwt% to gather
Sugar and the Ca of 0.05-0.1wt%2+、Mg2+、Zn2+On the PDA culture medium flat board of salt, each dilution is repeated twice, will be coated
Culture dish be inverted be put in 28 DEG C under the conditions of cultivate 48h, select coating culture after length have the flat board of Antagonistic Fungi standby;
By step (1) in the remaining botrytis cinerea PDA plate without picking in culture 5-7d to a large amount of spores generation after, use
0.5wt% Tween-80s wash lower botrytis cinerea Spores, obtain grey mold spore liquid, and blood counting chamber adjusts spore liquid miospore
Concentration is 104cfu/mL;
(3) the ratio according to 0.1-0.3wt% in the grey mold spore liquid that concentration is regulated adds N- acetyl glucosamines and chitin few
Sugar, against step (1) in the length selected have Antagonistic Fungi flat board fast spraying once, spray concentration is 104Cfu/mL botrytis cinerea spores
Sub- liquid, is inverted after 1h and is put in 28 DEG C of incubated 48h-72h, and the inhibition zone size after observation co-cultivation, according to the big of inhibition zone
Little screening Antagonistic Fungi, inhibition zone size are that inhibition zone external diameter subtracts the radius for detaining the Antagonistic Fungi for going to cultivate, and the bigger explanation of inhibition zone is short of money
Anti- effect is better;
(4) the bacterium with inhibition zone is carried out line purifying on nutrient medium flat board, until the bacterium colony on each flat board is presented
Identical color, size and shape, the Antagonistic Fungi for as purifying;
(5) secondary screening:By the Antagonistic Fungi dibbling for having purified on nutrient medium flat board, in 28 DEG C of incubators after culture 48h, spray ash
Continue culture 48h after mycotic spore liquid in 28 DEG C of incubators, observation have stable inhibition zone for botrytis cinerea Antagonistic Fungi.
2. high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi according to claim 1 quickly lures anti-screening technique, and its feature exists
In:Step nutrient medium (4) is wort agar culture medium or NYDA solid mediums;NYDA fluid nutrient mediums
Fill a prescription and be:Nutrient broth 8g, yeast extract 5.0g, glucose 10g, agar 18g, distilled water 1000mL, 121 DEG C of sterilizing 20min.
3. high biological and ecological methods to prevent plant disease, pests, and erosion power grape botrytis cinerea Antagonistic Fungi according to claim 1 quickly lures anti-screening technique, and its feature exists
In:Described nutrient medium is PDA liquid medium or PDA solid mediums;The formula of PDA liquid medium is:Ma Ling
Potato 300g, glucose 20g, chloramphenicol 0.1g, distilled water 1000mL, add agar 20g to form PDA in PDA liquid medium
Solid medium.
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CN114747614A (en) * | 2021-01-08 | 2022-07-15 | 天津科技大学 | Biological safe fresh-keeping paper for fruits and vegetables and preparation method thereof |
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CN107502560A (en) * | 2017-09-30 | 2017-12-22 | 中国科学院成都生物研究所 | A kind of concentration and separation technology of endophyte of plant and its application |
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CN114747614B (en) * | 2021-01-08 | 2024-05-24 | 天津科技大学 | Biological safety preservative paper for fruits and vegetables and preparation method thereof |
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