CN110317802A - A kind of microbial bacterial agent and preparation method thereof for preventing and treating ring rot of apple - Google Patents
A kind of microbial bacterial agent and preparation method thereof for preventing and treating ring rot of apple Download PDFInfo
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- CN110317802A CN110317802A CN201910473858.2A CN201910473858A CN110317802A CN 110317802 A CN110317802 A CN 110317802A CN 201910473858 A CN201910473858 A CN 201910473858A CN 110317802 A CN110317802 A CN 110317802A
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- 239000000843 powder Substances 0.000 claims abstract description 136
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 49
- 241000193755 Bacillus cereus Species 0.000 claims abstract description 39
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 37
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 37
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000000661 sodium alginate Substances 0.000 claims abstract description 35
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 35
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 35
- 241001465752 Purpureocillium lilacinum Species 0.000 claims abstract description 34
- 241001646398 Pseudomonas chlororaphis Species 0.000 claims abstract description 33
- 241000728344 Streptomyces hygrospinosus Species 0.000 claims abstract description 33
- PQUXFUBNSYCQAL-UHFFFAOYSA-N 1-(2,3-difluorophenyl)ethanone Chemical compound CC(=O)C1=CC=CC(F)=C1F PQUXFUBNSYCQAL-UHFFFAOYSA-N 0.000 claims abstract description 27
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 27
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
- USNWAMPROKAEIT-UHFFFAOYSA-N [Na].C(C=C)(=O)O Chemical compound [Na].C(C=C)(=O)O USNWAMPROKAEIT-UHFFFAOYSA-N 0.000 claims 1
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- 239000001888 Peptone Substances 0.000 description 24
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- 235000013399 edible fruits Nutrition 0.000 description 21
- 239000001963 growth medium Substances 0.000 description 17
- 229920001817 Agar Polymers 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000008272 agar Substances 0.000 description 16
- 235000013882 gravy Nutrition 0.000 description 16
- 239000002054 inoculum Substances 0.000 description 16
- 239000003643 water by type Substances 0.000 description 15
- CWXZAJNUTOBAOI-UHFFFAOYSA-N 1-(2,3-dimethoxyphenyl)-2-hydroxy-2-phenylethanone Chemical compound COC1=CC=CC(C(=O)C(O)C=2C=CC=CC=2)=C1OC CWXZAJNUTOBAOI-UHFFFAOYSA-N 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 9
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 8
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- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 7
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000015511 Liquidambar orientalis Nutrition 0.000 description 1
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- 239000004870 Styrax Substances 0.000 description 1
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- 235000000126 Styrax benzoin Nutrition 0.000 description 1
- 239000005842 Thiophanate-methyl Substances 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000006013 carbendazim Substances 0.000 description 1
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
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- 239000002207 metabolite Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical group C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
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- 238000003672 processing method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
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- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical compound COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- 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
- C12N1/14—Fungi; Culture media therefor
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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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- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of microbial bacterial agents and preparation method thereof for preventing and treating ring rot of apple, it is obtained using gel carrier load compound microorganism bacterium powder, compound microorganism bacterium powder includes: bacillus cereus ACCC03288 bacterium powder, Trichoderma harzianum ACCC30371 bacterium powder, Pseudomonas chlororaphis ACCC19853 bacterium powder, Paecilomyces lilacinus ACCC30639 bacterium powder, Streptomyces hygrospinosus ACCC40018 bacterium powder, collaboration inhibits the growth and breeding of Botryosphaeria berengeriana f. sp, and nutritional support is provided for apple tree, promote apple development.Gel carrier is the cross-linking reaction using acrylamide and sodium acrylate, and the crosslinking of sodium alginate and calcium chloride, and it introduces polyvinyl alcohol and obtains, it can natural degradation, slow releasing function is played to compound microorganism bacterium powder, and nutritional support is provided for microorganism growth, so that compound microorganism bacterium powder preferably plays preventive effect effect.Microbial bacterial agent first use of the invention, reduces labor intensity.
Description
Technical field
The present invention relates to technical fields, more particularly to a kind of microbial bacterial agent for preventing and treating ring rot of apple and its preparation side
Method.
Background technique
Ring spot is one of Common Diseases of apple, is to be infected to cause by weak parasite, including Trunk canker and fruit
Two kinds of ring spot.Ring spot germ is overwintering in killed limb with mycelia, pycnidia and the shell of ascus.Mycelia is in limb diseased tissues
In can survive 4~5 years, generate spore between annual 4~June, become primary infection source.7~August spore distributes more；Sick portion
The sporogenic ability of the first three years production is strong, gradually weakens later.Conidium is splashed depending mainly on rainwater propagates, and range is usually no more than
10 meters.Germ can infect young fruit in 10 days or so after fallen flowers.4~July, the amount of infecting was most, the scattered amount drop of spore after July
It is low, while fruit is also not easy to be infected, infection rate is very low.Spore germination percutaneous (fruit dot) intrusion fruit and limb, 24 hours can
Completion is infected.
Ring spot, Mancozeb, carbendazim, methyl Tobe are mainly prevented and treated by chemical bactericide in apple tree planting at present
The fungicide such as saliva, thiophanate-methyl, difoltan are that still these medical treatments are used for a long time in the common medicament of prevention and treatment ring spot
Ring rot of apple, it may appear that the problem of drug resistance and sensibility decline, and drug is easy residual, remaining pesticide can harmful to human
Health.
Microbial bacterial agent is rather well received in each field in recent years, utilizes the vital movement and metabolite of microorganism, changes
Kind crops nutrient supply provides nutritional support, prophyiaxis and promoting growth for crops, and resistance problems is not present.Patent
CN102805107B discloses a kind of microorganism formulation for preventing and treating ring rot of apple, be using bacillus subtilis activation,
Fermentation obtains, and control efficiency still has greatly improved space；And need multiple applications, including florescence April spray or
Trunk rough bark is smeared, fruit face is sprayed before June fruit bagging, seven August rainwater spray or smear processing trunk disease when more
Tissue, process is cumbersome, the large labor intensity of orchard worker.Having a major reason among these is after microbial bacterial agent is applied to environment ratio
More sensitive, application bad environments likely result in microorganism mortality, and then cause preventive effect to be deteriorated and be even reduced to zero.
Summary of the invention
Present invention aim to provide a kind of microbial bacterial agent and preparation method thereof for preventing and treating ring rot of apple.
To achieve the above object, the present invention is achieved by the following scheme:
A kind of microbial bacterial agent for preventing and treating ring rot of apple, including compound microorganism bacterium powder and the gel carrier for loading it
Two parts, in parts by weight, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, breathing out thatch wood
Mould 0.2~0.3 part of ACCC30371 bacterium powder, 0.3~0.5 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus
0.1~0.2 part of ACCC30639 bacterium powder, 0.3~0.5 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carrier is
It is prepared by the following method: acrylamide and sodium acrylate being first added into polyvinyl alcohol water solution, passes through ultraviolet photograph
It penetrates reaction and obtains gel intermediate, then gel intermediate is soaked in sodium alginate soln, instill calcium chloride solution, crosslinking
Reaction, closing are placed, and washing obtains gel carrier.
Preferably, the mass ratio of compound microorganism bacterium powder and gel carrier is 1:20~30.
Preferably, the compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus
ACCC03288, Trichoderma harzianum ACCC30371, Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, thorn spore are inhaled
Water streptomycete ACCC40018 bacterial strain individually carries out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtains corresponding bacterium
Then powder mixes gained bacterium powder to get the compound microorganism bacterium powder.
It is further preferred that bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone
Culture medium, inoculum concentration are 4~5% (volumes)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, thorn spore water suction chain
Mould ACCC40018 strain inoculated is in PDA culture medium, and inoculum concentration is 3~4% (volumes), after culture 4~6 days, respectively at 170
~180r/min shaking table culture, respective condition of culture are as follows: the cultivation temperature of bacillus cereus ACCC03288 is 27~29
DEG C, incubation time is 40~45 hours；The cultivation temperature of Pseudomonas chlororaphis ACCC19853 is 27~29 DEG C, and incubation time is
42~46 hours；The cultivation temperature of bacillus coagulans ACCC00403 is 28~30 DEG C, and incubation time is 45~48 hours；It breathes out
The cultivation temperature of thatch trichoderma ACCC30423 is 27~29 DEG C, and incubation time is 72~75 hours；Paecilomyces lilacinus ACCC30639
Cultivation temperature be 28~30 DEG C, incubation time be 58~62 hours；The cultivation temperature of Streptomyces hygrospinosus ACCC40018 is
26~28 DEG C, incubation time is 55~60 hours.
It is further preferred that in parts by weight, the gravy peptone culture mediums include: 3~5 parts of beef extract, peptone 5~6
Part, 2~4 parts of NaCl, 18~22 parts and 1000 parts deionized waters of agar, and its pH is 6.8~7.2；In parts by weight, described
PDA culture medium includes: 195~200 parts of potato, 10~12 parts of glucose, 18~20 parts of agar, Na2HPO44~6 parts and 1000 parts
Deionized water, and its pH is 7.0~7.6.
It is further preferred that being flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively inoculated and cultured institute
Obtain the 1~2% and 0.5~0.8% of quality of fermentation broth.
It is further preferred that filters pressing, drying and crushing method particularly includes: plate compression is used, is then done for 45~50 DEG C
It is dry to be crushed using pulverizer after cyclonic separation to water content 20~30%, 30 meshes are crossed to get the bacterium powder.
Preferably, the gel carrier is prepared by the following method to obtain: being first added into polyvinyl alcohol water solution
Acrylamide and sodium acrylate, add crosslinking agent and photoinitiator is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 30~40 minutes, seal
It closes placement 10~12 hours, washs, obtain gel carrier.
It is further preferred that after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, wherein logical nitrogen removes
The specific method of oxygen is: nitrogen is bubbled 10~30 minutes.
It is further preferred that polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, alginic acid
Sodium solution, calcium chloride solution mass ratio be 1:0.2~0.3:1.5~1.8:0.1~0.2:0.03~0.04:1~2:6~8,
Polyvinyl alcohol water solution, sodium alginate soln, calcium chloride solution mass concentration be followed successively by 5~8%, 2~3%, 2~3%.
It is further preferred that the crosslinking agent is N, N '-methylene-bisacrylamide, photoinitiator is styrax diformazan
Ether, dimethoxybenzoin are dissolved in the solution use that mass concentration 2.5% is configured in N-Methyl pyrrolidone.
It is further preferred that the process conditions of ultraviolet irradiation reaction are as follows: the ultraviolet light irradiation of wavelength 365nm, power 400W
Reaction 5~7 hours.
It is further preferred that the specific method of washing is: benefit is washed with deionized 3~5 times, to clean remaining reaction
Reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs by a kind of preparation method of above-mentioned microbial bacterial agent for preventing and treating ring rot of apple
It is even to obtain aqueous solution, then gel carrier is poured into aqueous solution, with 10~15L/ minutes air velocity blowing airs, 1000~
1500 revs/min are stirred 30~40 minutes, and closing stands 5~8 hours, dry to get the microbial bacterial agent.
Preferably, the mass ratio of microbial bacterial agent and water is 1:8~10.
Preferably, dry process conditions are as follows: (25 DEG C) of room temperature are dried in vacuo 12~15 hours.
Application of the mentioned microorganism microbial inoculum in prevention and treatment ring rot of apple.
Microbial bacterial agent is added to dilute in the water of 100 times of weight and dissolve, then by the application method of mentioned microorganism microbial inoculum
It is applied to trunk rough bark surface, applying amount is 0.3~0.5mL/cm2。
The beneficial effects of the present invention are:
The present invention obtains a kind of microbial bacterial agent, compound microorganism bacterium powder using gel carrier load compound microorganism bacterium powder
Include: bacillus cereus ACCC03288 bacterium powder, Trichoderma harzianum ACCC30371 bacterium powder, Pseudomonas chlororaphis ACCC19853 bacterium
Powder, Paecilomyces lilacinus ACCC30639 bacterium powder, Streptomyces hygrospinosus ACCC40018 bacterium powder, collaboration inhibit Botryosphaeria berengeriana f. sp
Growth and breeding, and nutritional support is provided for apple tree, promote apple development.Gel carrier is to utilize acrylamide and sodium acrylate
Cross-linking reaction and sodium alginate and calcium chloride crosslinking, and introduce polyvinyl alcohol and obtain, can natural degradation, to compound micro-
Biological bacteria powder plays slow releasing function, and provides nutritional support for microorganism growth, so that compound microorganism bacterium powder preferably plays
Preventive effect effect.Microbial bacterial agent first use of the invention, reduces labor intensity.
Bacillus cereus ACCC03288 can produce antibacterial material, inhibit the breeding of Target spot pathogen；Trichoderma harzianum
ACCC30371 growth rapidly, can seize trunk surface site, form a protective cover, to protect apple tree from taking turns line
Germ is infected；Pseudomonas chlororaphis ACCC19853 has very strong inhibiting effect to Target spot pathogen, and can generate active material
Promote the growth of apple tree；Paecilomyces lilacinus ACCC30639 breeding quickly, can secrete a variety of organic acids of synthesis, enzyme, physiological activity
Substance inhibits the growth and breeding of Target spot pathogen；Streptomyces hygrospinosus ACCC40018 generates antibacterial material, inhibits Target spot pathogen
Growth and breeding.They cooperate with the growth and breeding for inhibiting Botryosphaeria berengeriana f. sp, and ring rot of apple preventive effect is high, and provides for apple tree
Nutritional support promotes apple development.
It with the cross-linking reaction of acrylamide and sodium acrylate, is also wound with polyvinyl alcohol, forms gel intermediate,
Then it being soaked in sodium alginate soln, instills calcium chloride solution, sodium alginate, which encounters calcium ion, can occur rapidly ion exchange,
Further gelation forms solid netted gel carrier.Under the load effect of gel carrier, enhancing microbial bacterial agent is resisted
The ability of poor environment guarantees microbial activity, promotes microbial reproduction growth.Polyacrylamide, polyvinyl alcohol etc. can be micro-
Biology provides nutrient source, can convert small molecule for polyacrylamide, polyvinyl alcohol etc. again during microbial growth and metabolism
Organic matter and inorganic matter realize natural degradation.Microorganism in compound microorganism bacterium powder from gel carrier slowly, sustained release,
Effectively extend action time, ring rot of apple preventive effect is high.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Bacillus cereus ACCC03288 of the present invention, Trichoderma harzianum ACCC30371, Pseudomonas chlororaphis
ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018, are purchased from Chinese agriculture microbial bacteria
Kind preservation administrative center.
Embodiment 1
A kind of microbial bacterial agent for preventing and treating ring rot of apple, including compound microorganism bacterium powder and the gel carrier for loading it
Two parts, in parts by weight, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, breathing out thatch wood
Mould 0.2 part of ACCC30371 bacterium powder, 0.3 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus ACCC30639 bacterium powder
0.1 part, 0.3 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carrier is prepared by the following method to obtain:
Acrylamide and sodium acrylate are first added into polyvinyl alcohol water solution, reacts to obtain gel intermediate by ultraviolet irradiation, so
Gel intermediate is soaked in sodium alginate soln afterwards, instills calcium chloride solution, cross-linking reaction, closing is placed, and washing obtains
Gel carrier.
The mass ratio of compound microorganism bacterium powder and gel carrier is 1:20.
Compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus ACCC03288, Ha Ci
Trichoderma ACCC30371, Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus
ACCC40018 bacterial strain individually carries out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtains corresponding bacterium powder, then will
Gained bacterium powder is mixed to get the compound microorganism bacterium powder.
Bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone culture mediums, inoculum concentration
For 4% (volume)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterial strain
It is inoculated in PDA culture medium, inoculum concentration is 3% (volume), after culture 4 days, respectively at 170r/min shaking table culture, respective culture
Condition is as follows: the cultivation temperature of bacillus cereus ACCC03288 is 27 DEG C, and incubation time is 40 hours；Pseudomonas chlororaphis
The cultivation temperature of ACCC19853 is 27 DEG C, and incubation time is 42 hours；The cultivation temperature of bacillus coagulans ACCC00403 is
28 DEG C, incubation time is 45 hours；The cultivation temperature of Trichoderma harzianum ACCC30423 is 27 DEG C, and incubation time is 72 hours；It is pale purple
The cultivation temperature of Paecilomyces varioti ACCC30639 is 28 DEG C, and incubation time is 58 hours；The training of Streptomyces hygrospinosus ACCC40018
Supporting temperature is 26 DEG C, and incubation time is 55 hours.
In parts by weight, gravy peptone culture mediums include: 3 parts of beef extract, 5 parts of peptone, 2 parts of NaCl, 18 parts of agar and
1000 parts of deionized waters, and its pH is 6.8；In parts by weight, PDA culture medium includes: 195 parts of potato, 10 parts of glucose, agar
18 parts, Na2HPO44 parts and 1000 parts of deionized waters, and its pH is 7.0.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
1% and 0.5%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 45 DEG C of dryings to water content 20%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Gel carrier is prepared by the following method to obtain: acrylamide and third being first added into polyvinyl alcohol water solution
Olefin(e) acid sodium, after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 30 minutes, closing was put
It sets 10 hours, washs, obtain gel carrier.Wherein, the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 10 minutes.
Polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride
The mass ratio of solution is 1:0.2:1.5:0.1:0.03:1:6, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride solution
Mass concentration is followed successively by 5%, 2%, 2%.
Crosslinking agent is N, and N '-methylene-bisacrylamide, photoinitiator is dimethoxybenzoin, and dimethoxybenzoin is molten
The solution that solution is configured to mass concentration 2.5% in N-Methyl pyrrolidone uses.The process conditions of ultraviolet irradiation reaction are as follows: wave
Ultraviolet light irradiation reaction 5 hours of long 365nm, power 400W.The specific method of washing is: benefit is washed with deionized 3 times, with
Clean remaining reaction reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs by a kind of preparation method of above-mentioned microbial bacterial agent for preventing and treating ring rot of apple
It is even to obtain aqueous solution, then gel carrier is poured into aqueous solution, with 10L/ minutes air velocity blowing airs, 1000 revs/min
Clock stirs 30 minutes, and closing stands 5 hours, dry to get the microbial bacterial agent.The mass ratio of microbial bacterial agent and water is
1:8.Dry process conditions are as follows: (25 DEG C) of room temperature are dried in vacuo 12 hours.
Embodiment 2
A kind of microbial bacterial agent for preventing and treating ring rot of apple, including compound microorganism bacterium powder and the gel carrier for loading it
Two parts, in parts by weight, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, breathing out thatch wood
Mould 0.3 part of ACCC30371 bacterium powder, 0.5 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus ACCC30639 bacterium powder
0.2 part, 0.5 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carrier is prepared by the following method to obtain:
Acrylamide and sodium acrylate are first added into polyvinyl alcohol water solution, reacts to obtain gel intermediate by ultraviolet irradiation, so
Gel intermediate is soaked in sodium alginate soln afterwards, instills calcium chloride solution, cross-linking reaction, closing is placed, and washing obtains
Gel carrier.
The mass ratio of compound microorganism bacterium powder and gel carrier is 1:30.
Compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus ACCC03288, Ha Ci
Trichoderma ACCC30371, Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus
ACCC40018 bacterial strain individually carries out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtains corresponding bacterium powder, then will
Gained bacterium powder is mixed to get the compound microorganism bacterium powder.
Bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone culture mediums, inoculum concentration
For 5% (volume)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterial strain
It is inoculated in PDA culture medium, inoculum concentration is 4% (volume), after culture 6 days, respectively at 180r/min shaking table culture, respective culture
Condition is as follows: the cultivation temperature of bacillus cereus ACCC03288 is 29 DEG C, and incubation time is 45 hours；Pseudomonas chlororaphis
The cultivation temperature of ACCC19853 is 29 DEG C, and incubation time is 46 hours；The cultivation temperature of bacillus coagulans ACCC00403 is
30 DEG C, incubation time is 48 hours；The cultivation temperature of Trichoderma harzianum ACCC30423 is 29 DEG C, and incubation time is 75 hours；It is pale purple
The cultivation temperature of Paecilomyces varioti ACCC30639 is 30 DEG C, and incubation time is 62 hours；The training of Streptomyces hygrospinosus ACCC40018
Supporting temperature is 28 DEG C, and incubation time is 60 hours.
In parts by weight, gravy peptone culture mediums include: 5 parts of beef extract, 6 parts of peptone, 4 parts of NaCl, 22 parts of agar and
1000 parts of deionized waters, and its pH is 7.2；In parts by weight, PDA culture medium includes: 200 parts of potato, 12 parts of glucose, agar
20 parts, Na2HPO46 parts and 1000 parts of deionized waters, and its pH is 7.6.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
2% and 0.8%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 50 DEG C of dryings to water content 30%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Gel carrier is prepared by the following method to obtain: acrylamide and third being first added into polyvinyl alcohol water solution
Olefin(e) acid sodium, after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 40 minutes, closing was put
It sets 12 hours, washs, obtain gel carrier.Wherein, the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 30 minutes.
Polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride
The mass ratio of solution is 1:0.3:1.8:0.2:0.04:2:8, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride solution
Mass concentration is followed successively by 8%, 3%, 3%.
Crosslinking agent is N, and N '-methylene-bisacrylamide, photoinitiator is dimethoxybenzoin, and dimethoxybenzoin is molten
The solution that solution is configured to mass concentration 2.5% in N-Methyl pyrrolidone uses.The process conditions of ultraviolet irradiation reaction are as follows: wave
Ultraviolet light irradiation reaction 7 hours of long 365nm, power 400W.The specific method of washing is: benefit is washed with deionized 5 times, with
Clean remaining reaction reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs by a kind of preparation method of above-mentioned microbial bacterial agent for preventing and treating ring rot of apple
It is even to obtain aqueous solution, then gel carrier is poured into aqueous solution, with 15L/ minutes air velocity blowing airs, 1500 revs/min
Clock stirs 40 minutes, and closing stands 8 hours, dry to get the microbial bacterial agent.The mass ratio of microbial bacterial agent and water is
1:10.Dry process conditions are as follows: (25 DEG C) of room temperature are dried in vacuo 15 hours.
Embodiment 3
A kind of microbial bacterial agent for preventing and treating ring rot of apple, including compound microorganism bacterium powder and the gel carrier for loading it
Two parts, in parts by weight, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, breathing out thatch wood
Mould 0.2 part of ACCC30371 bacterium powder, 0.5 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus ACCC30639 bacterium powder
0.1 part, 0.3 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carrier is prepared by the following method to obtain:
Acrylamide and sodium acrylate are first added into polyvinyl alcohol water solution, reacts to obtain gel intermediate by ultraviolet irradiation, so
Gel intermediate is soaked in sodium alginate soln afterwards, instills calcium chloride solution, cross-linking reaction, closing is placed, and washing obtains
Gel carrier.
The mass ratio of compound microorganism bacterium powder and gel carrier is 1:30.
Compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus ACCC03288, Ha Ci
Trichoderma ACCC30371, Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus
ACCC40018 bacterial strain individually carries out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtains corresponding bacterium powder, then will
Gained bacterium powder is mixed to get the compound microorganism bacterium powder.
Bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone culture mediums, inoculum concentration
For 4% (volume)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterial strain
It is inoculated in PDA culture medium, inoculum concentration is 4% (volume), after culture 4 days, respectively at 180r/min shaking table culture, respective culture
Condition is as follows: the cultivation temperature of bacillus cereus ACCC03288 is 27 DEG C, and incubation time is 45 hours；Pseudomonas chlororaphis
The cultivation temperature of ACCC19853 is 27 DEG C, and incubation time is 46 hours；The cultivation temperature of bacillus coagulans ACCC00403 is
28 DEG C, incubation time is 48 hours；The cultivation temperature of Trichoderma harzianum ACCC30423 is 27 DEG C, and incubation time is 75 hours；It is pale purple
The cultivation temperature of Paecilomyces varioti ACCC30639 is 28 DEG C, and incubation time is 62 hours；The training of Streptomyces hygrospinosus ACCC40018
Supporting temperature is 26 DEG C, and incubation time is 60 hours.
In parts by weight, gravy peptone culture mediums include: 3 parts of beef extract, 6 parts of peptone, 2 parts of NaCl, 22 parts of agar and
1000 parts of deionized waters, and its pH is 6.8；In parts by weight, PDA culture medium includes: 200 parts of potato, 10 parts of glucose, agar
20 parts, Na2HPO44 parts and 1000 parts of deionized waters, and its pH is 7.6.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
1% and 0.8%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 45 DEG C of dryings to water content 30%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Gel carrier is prepared by the following method to obtain: acrylamide and third being first added into polyvinyl alcohol water solution
Olefin(e) acid sodium, after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 30 minutes, closing was put
It sets 12 hours, washs, obtain gel carrier.Wherein, the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 10 minutes.
Polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride
The mass ratio of solution is 1:0.3:1.5:0.2:0.03:2:6, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride solution
Mass concentration is followed successively by 8%, 2%, 3%.
Crosslinking agent is N, and N '-methylene-bisacrylamide, photoinitiator is dimethoxybenzoin, and dimethoxybenzoin is molten
The solution that solution is configured to mass concentration 2.5% in N-Methyl pyrrolidone uses.The process conditions of ultraviolet irradiation reaction are as follows: wave
Ultraviolet light irradiation reaction 5 hours of long 365nm, power 400W.The specific method of washing is: benefit is washed with deionized 5 times, with
Clean remaining reaction reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs by a kind of preparation method of above-mentioned microbial bacterial agent for preventing and treating ring rot of apple
It is even to obtain aqueous solution, then gel carrier is poured into aqueous solution, with 10L/ minutes air velocity blowing airs, 1500 revs/min
Clock stirs 30 minutes, and closing stands 8 hours, dry to get the microbial bacterial agent.The mass ratio of microbial bacterial agent and water is
1:8.Dry process conditions are as follows: (25 DEG C) of room temperature are dried in vacuo 15 hours.
Embodiment 4
A kind of microbial bacterial agent for preventing and treating ring rot of apple, including compound microorganism bacterium powder and the gel carrier for loading it
Two parts, in parts by weight, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, breathing out thatch wood
Mould 0.3 part of ACCC30371 bacterium powder, 0.3 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus ACCC30639 bacterium powder
0.2 part, 0.5 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carrier is prepared by the following method to obtain:
Acrylamide and sodium acrylate are first added into polyvinyl alcohol water solution, reacts to obtain gel intermediate by ultraviolet irradiation, so
Gel intermediate is soaked in sodium alginate soln afterwards, instills calcium chloride solution, cross-linking reaction, closing is placed, and washing obtains
Gel carrier.
The mass ratio of compound microorganism bacterium powder and gel carrier is 1:20.
Compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus ACCC03288, Ha Ci
Trichoderma ACCC30371, Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus
ACCC40018 bacterial strain individually carries out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtains corresponding bacterium powder, then will
Gained bacterium powder is mixed to get the compound microorganism bacterium powder.
Bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone culture mediums, inoculum concentration
For 5% (volume)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterial strain
It is inoculated in PDA culture medium, inoculum concentration is 3% (volume), after culture 6 days, respectively at 170r/min shaking table culture, respective culture
Condition is as follows: the cultivation temperature of bacillus cereus ACCC03288 is 29 DEG C, and incubation time is 40 hours；Pseudomonas chlororaphis
The cultivation temperature of ACCC19853 is 29 DEG C, and incubation time is 42 hours；The cultivation temperature of bacillus coagulans ACCC00403 is
30 DEG C, incubation time is 45 hours；The cultivation temperature of Trichoderma harzianum ACCC30423 is 29 DEG C, and incubation time is 72 hours；It is pale purple
The cultivation temperature of Paecilomyces varioti ACCC30639 is 30 DEG C, and incubation time is 58 hours；The training of Streptomyces hygrospinosus ACCC40018
Supporting temperature is 28 DEG C, and incubation time is 55 hours.
In parts by weight, gravy peptone culture mediums include: 5 parts of beef extract, 5 parts of peptone, 4 parts of NaCl, 18 parts of agar and
1000 parts of deionized waters, and its pH is 7.2；In parts by weight, PDA culture medium includes: 195 parts of potato, 12 parts of glucose, agar
18 parts, Na2HPO46 parts and 1000 parts of deionized waters, and its pH is 7.0.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
2% and 0.5%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 50 DEG C of dryings to water content 20%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Gel carrier is prepared by the following method to obtain: acrylamide and third being first added into polyvinyl alcohol water solution
Olefin(e) acid sodium, after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 40 minutes, closing was put
It sets 10 hours, washs, obtain gel carrier.Wherein, the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 30 minutes.
Polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride
The mass ratio of solution is 1:0.2:1.8:0.1:0.04:1:8, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride solution
Mass concentration is followed successively by 5%, 3%, 2%.
Crosslinking agent is N, and N '-methylene-bisacrylamide, photoinitiator is dimethoxybenzoin, and dimethoxybenzoin is molten
The solution that solution is configured to mass concentration 2.5% in N-Methyl pyrrolidone uses.The process conditions of ultraviolet irradiation reaction are as follows: wave
Ultraviolet light irradiation reaction 7 hours of long 365nm, power 400W.The specific method of washing is: benefit is washed with deionized 3 times, with
Clean remaining reaction reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs by a kind of preparation method of above-mentioned microbial bacterial agent for preventing and treating ring rot of apple
It is even to obtain aqueous solution, then gel carrier is poured into aqueous solution, with 15L/ minutes air velocity blowing airs, 1000 revs/min
Clock stirs 40 minutes, and closing stands 5 hours, dry to get the microbial bacterial agent.The mass ratio of microbial bacterial agent and water is
1:10.Dry process conditions are as follows: (25 DEG C) of room temperature are dried in vacuo 12 hours.
Embodiment 5
A kind of microbial bacterial agent for preventing and treating ring rot of apple, including compound microorganism bacterium powder and the gel carrier for loading it
Two parts, in parts by weight, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, breathing out thatch wood
Mould 0.25 part of ACCC30371 bacterium powder, 0.4 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus ACCC30639 bacterium powder
0.15 part, 0.4 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carrier is prepared by the following method to obtain:
Acrylamide and sodium acrylate are first added into polyvinyl alcohol water solution, reacts to obtain gel intermediate by ultraviolet irradiation, so
Gel intermediate is soaked in sodium alginate soln afterwards, instills calcium chloride solution, cross-linking reaction, closing is placed, and washing obtains
Gel carrier.
The mass ratio of compound microorganism bacterium powder and gel carrier is 1:25.
Compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus ACCC03288, Ha Ci
Trichoderma ACCC30371, Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus
ACCC40018 bacterial strain individually carries out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtains corresponding bacterium powder, then will
Gained bacterium powder is mixed to get the compound microorganism bacterium powder.
Bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone culture mediums, inoculum concentration
For 4.5% (volume)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterium
Strain is inoculated in PDA culture medium, and inoculum concentration is 3.5% (volume), respective respectively at 170r/min shaking table culture after culture 5 days
Condition of culture is as follows: the cultivation temperature of bacillus cereus ACCC03288 is 28 DEG C, and incubation time is 42 hours；Green needle is false single
The cultivation temperature of born of the same parents bacterium ACCC19853 is 28 DEG C, and incubation time is 44 hours；The culture temperature of bacillus coagulans ACCC00403
Degree is 29 DEG C, and incubation time is 46 hours；The cultivation temperature of Trichoderma harzianum ACCC30423 is 28 DEG C, and incubation time is 74 hours；
The cultivation temperature of Paecilomyces lilacinus ACCC30639 is 29 DEG C, and incubation time is 60 hours；Streptomyces hygrospinosus ACCC40018
Cultivation temperature be 27 DEG C, incubation time be 58 hours.
In parts by weight, gravy peptone culture mediums include: 4 parts of beef extract, 5 parts of peptone, 3 parts of NaCl, 20 parts of agar and
1000 parts of deionized waters, and its pH is 7；In parts by weight, PDA culture medium includes: 198 parts of potato, 11 parts of glucose, agar 19
Part, Na2HPO45 parts and 1000 parts of deionized waters, and its pH is 7.2.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
1.5% and 0.7%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 48 DEG C of dryings to water content 25%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Gel carrier is prepared by the following method to obtain: acrylamide and third being first added into polyvinyl alcohol water solution
Olefin(e) acid sodium, after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 35 minutes, closing was put
It sets 11 hours, washs, obtain gel carrier.Wherein, the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 20 minutes.
Polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride
The mass ratio of solution is 1:0.25:1.6:0.15:0.035:1.5:7, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride
The mass concentration of solution is followed successively by 6%, 2.5%, 2.5%.
Crosslinking agent is N, and N '-methylene-bisacrylamide, photoinitiator is dimethoxybenzoin, and dimethoxybenzoin is molten
The solution that solution is configured to mass concentration 2.5% in N-Methyl pyrrolidone uses.The process conditions of ultraviolet irradiation reaction are as follows: wave
Ultraviolet light irradiation reaction 6 hours of long 365nm, power 400W.The specific method of washing is: benefit is washed with deionized 4 times, with
Clean remaining reaction reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs by a kind of preparation method of above-mentioned microbial bacterial agent for preventing and treating ring rot of apple
It is even to obtain aqueous solution, then gel carrier is poured into aqueous solution, with 12L/ minutes air velocity blowing airs, 1200 revs/min
Clock stirs 35 minutes, and closing stands 6 hours, dry to get the microbial bacterial agent.The mass ratio of microbial bacterial agent and water is
1:9.Dry process conditions are as follows: (25 DEG C) of room temperature are dried in vacuo 13 hours.
Comparative example 1
A kind of microbial bacterial agent, including compound microorganism bacterium powder and the gel carrier two parts for loading it, with parts by weight
Meter, the compound microorganism bacterium powder includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, Trichoderma harzianum ACCC30371 bacterium powder
0.25 part, 0.15 part of powder of Paecilomyces lilacinus ACCC30639 bacterium；The gel carrier is prepared by the following method to obtain: first
Acrylamide and sodium acrylate are added into polyvinyl alcohol water solution, reacts to obtain gel intermediate by ultraviolet irradiation, then
Gel intermediate is soaked in sodium alginate soln, calcium chloride solution, cross-linking reaction are instilled, closing is placed, and washing is coagulated
Glue carrier.
The mass ratio of compound microorganism bacterium powder and gel carrier is 1:25.
Compound microorganism bacterium powder is prepared by the following preparation method: first by bacillus cereus ACCC03288, Ha Ci
Trichoderma ACCC30371, Paecilomyces lilacinus ACCC30639 bacterial strain individually carry out inoculated and cultured, flocculation, filters pressing, drying and powder
It is broken, corresponding bacterium powder is obtained, then mixes gained bacterium powder to get the compound microorganism bacterium powder.
Bacillus cereus ACCC03288 is inoculated in gravy peptone culture mediums, and inoculum concentration is 4.5% (volume)；Trichoderma harzianum
In PDA culture medium, inoculum concentration is 3.5% (volume) for ACCC30371, Paecilomyces lilacinus ACCC30639 strain inoculated, is cultivated 5 days
Afterwards, respectively at 170r/min shaking table culture, respective condition of culture is as follows: the cultivation temperature of bacillus cereus ACCC03288
It is 28 DEG C, incubation time is 42 hours；The cultivation temperature of Pseudomonas chlororaphis ACCC19853 is 28 DEG C, and incubation time is 44 small
When；The cultivation temperature of bacillus coagulans ACCC00403 is 29 DEG C, and incubation time is 46 hours；Trichoderma harzianum ACCC30423's
Cultivation temperature is 28 DEG C, and incubation time is 74 hours；The cultivation temperature of Paecilomyces lilacinus ACCC30639 is 29 DEG C, incubation time
It is 60 hours；The cultivation temperature of Streptomyces hygrospinosus ACCC40018 is 27 DEG C, and incubation time is 58 hours.
In parts by weight, gravy peptone culture mediums include: 4 parts of beef extract, 5 parts of peptone, 3 parts of NaCl, 20 parts of agar and
1000 parts of deionized waters, and its pH is 7；In parts by weight, PDA culture medium includes: 198 parts of potato, 11 parts of glucose, agar 19
Part, Na2HPO45 parts and 1000 parts of deionized waters, and its pH is 7.2.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
1.5% and 0.7%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 48 DEG C of dryings to water content 25%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Gel carrier is prepared by the following method to obtain: acrylamide and third being first added into polyvinyl alcohol water solution
Olefin(e) acid sodium, after crosslinking agent is added, letting nitrogen in and deoxidizing adds photoinitiator and is uniformly mixed, and ultraviolet irradiation reaction obtains among gel
Then gel intermediate is soaked in sodium alginate soln by body, instill calcium chloride solution, cross-linking reaction 35 minutes, closing was put
It sets 11 hours, washs, obtain gel carrier.Wherein, the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 20 minutes.
Polyvinyl alcohol water solution, acrylamide, sodium acrylate, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride
The mass ratio of solution is 1:0.25:1.6:0.15:0.035:1.5:7, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride
The mass concentration of solution is followed successively by 6%, 2.5%, 2.5%.
Crosslinking agent is N, and N '-methylene-bisacrylamide, photoinitiator is dimethoxybenzoin, and dimethoxybenzoin is molten
The solution that solution is configured to mass concentration 2.5% in N-Methyl pyrrolidone uses.The process conditions of ultraviolet irradiation reaction are as follows: wave
Ultraviolet light irradiation reaction 6 hours of long 365nm, power 400W.The specific method of washing is: benefit is washed with deionized 4 times, with
Clean remaining reaction reagent (sodium acrylate, calcium chloride etc.).
Microbial bacterial agent, is first added to the water and stirs evenly to obtain aqueous solution, so by a kind of preparation method of above-mentioned microbial bacterial agent
Gel carrier is poured into aqueous solution afterwards, with 12L/ minutes air velocity blowing airs, 1200 revs/min were stirred 35 minutes, envelope
It closes, stands 6 hours, it is dry to get the microbial bacterial agent.The mass ratio of microbial bacterial agent and water is 1:9.Dry technique item
Part are as follows: (25 DEG C) of room temperature are dried in vacuo 13 hours.
Comparative example 2
A kind of microbial bacterial agent includes in parts by weight: 1 part of powder of bacillus cereus ACCC03288 bacterium, Trichoderma harzianum
0.25 part of ACCC30371 bacterium powder, 0.4 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, Paecilomyces lilacinus ACCC30639 bacterium powder
0.15 part, 0.25 part of powder of streptomycete ACCC19748 bacterium of Lou Che Shi, 0.4 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium.
Be prepared by the following preparation method: first by bacillus cereus ACCC03288, Trichoderma harzianum ACCC30371,
Pseudomonas chlororaphis ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterial strain are individually
Carry out inoculated and cultured, flocculation, filters pressing, drying and crushing, obtain corresponding bacterium powder, then by gained bacterium powder mix to get
The compound microorganism bacterium powder.
Bacillus cereus ACCC03288, Pseudomonas chlororaphis ACCC19853 are inoculated in gravy peptone culture mediums, inoculum concentration
For 4.5% (volume)；Trichoderma harzianum ACCC30371, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterium
Strain is inoculated in PDA culture medium, and inoculum concentration is 3.5% (volume), respective respectively at 170r/min shaking table culture after culture 5 days
Condition of culture is as follows: the cultivation temperature of bacillus cereus ACCC03288 is 28 DEG C, and incubation time is 42 hours；Green needle is false single
The cultivation temperature of born of the same parents bacterium ACCC19853 is 28 DEG C, and incubation time is 44 hours；The culture temperature of bacillus coagulans ACCC00403
Degree is 29 DEG C, and incubation time is 46 hours；The cultivation temperature of Trichoderma harzianum ACCC30423 is 28 DEG C, and incubation time is 74 hours；
The cultivation temperature of Paecilomyces lilacinus ACCC30639 is 29 DEG C, and incubation time is 60 hours；Streptomyces hygrospinosus ACCC40018
Cultivation temperature be 27 DEG C, incubation time be 58 hours.
In parts by weight, gravy peptone culture mediums include: 4 parts of beef extract, 5 parts of peptone, 3 parts of NaCl, 20 parts of agar and
1000 parts of deionized waters, and its pH is 7；In parts by weight, PDA culture medium includes: 198 parts of potato, 11 parts of glucose, agar 19
Part, Na2HPO45 parts and 1000 parts of deionized waters, and its pH is 7.2.
It is flocculated using disodium hydrogen phosphate and calcium chloride, the additional amount of the two is respectively quality of fermentation broth obtained by inoculated and cultured
1.5% and 0.7%.
Filters pressing, drying and crushing method particularly includes: use plate compression, then 48 DEG C of dryings to water content 25%, rotation
It is crushed after wind separation using pulverizer, crosses 30 meshes to get the bacterium powder.
Test example
Time: in mid-April, 2018
Place: Zibo City, Shandong Province, the town Zhong Zhuan, Yiyuan County
Test variety: Fuji apple, the age of tree 10 years
Randomly select 2017 annual ring veins diseases fall ill 80 plants of fruit tree, be randomly divided into 8 groups, wherein one group as a control group, not into
Any processing of row, remaining 7 groups are used as processing group, are utilized respectively at the microbial bacterial agent of Examples 1 to 5 or comparative example 1~2
Reason, processing method are as follows: in fruit tree florescence in April, microbial bacterial agent is added to dilute in the water of 100 times of weight and is dissolved, then
It is applied to trunk rough bark surface, applying amount 0.4mL/cm2.Picking time diseased fruit rate (there is scab on surface) is counted after ripe apples,
And control efficiency, and choose fruit (surface disease-free spot) room temperature storage 15 days, diseased fruit rate (there is scab on surface) after statistics 15 days,
And control efficiency, it the results are shown in Table 1.
Diseased fruit rate=disease fruit/fruit sum × 100%；
Control efficiency=(control group diseased fruit rate-processing group diseased fruit rate)/control group diseased fruit rate × 100%.
1. control efficiency of table compares
As shown in Table 1, compared with the control group, prevention and treatment of the microbial bacterial agent of Examples 1 to 5 in picking time and storage period is imitated
Fruit is very excellent.Compound microorganism bacterium powder in comparative example 1 includes: 1 part of powder of bacillus cereus ACCC03288 bacterium, Ha Ci
0.25 part of powder of trichoderma ACCC30371 bacterium, 0.15 part of powder of Paecilomyces lilacinus ACCC30639 bacterium, reduce the collaboration between each bacterium powder
Synergistic effect, diseased fruit rate is significantly raised, and control efficiency is obviously deteriorated；Comparative example 2 directly uses compound microorganism bacterium powder as micro- life
Object microbial inoculum cannot achieve long term, and diseased fruit rate is significantly raised, and control efficiency is obviously deteriorated.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (9)
1. a kind of microbial bacterial agent for preventing and treating ring rot of apple, which is characterized in that including compound microorganism bacterium powder and load it
Gel carrier two parts, in parts by weight, the compound microorganism bacterium powder includes: bacillus cereus ACCC03288 bacterium powder 1
Part, it is 0.2~0.3 part of powder of Trichoderma harzianum ACCC30371 bacterium, 0.3~0.5 part of powder of Pseudomonas chlororaphis ACCC19853 bacterium, pale purple quasi-
0.1~0.2 part of powder of mould ACCC30639 bacterium, 0.3~0.5 part of powder of Streptomyces hygrospinosus ACCC40018 bacterium；The gel carries
Body is prepared by the following method to obtain: acrylamide and sodium acrylate being first added into polyvinyl alcohol water solution, passes through purple
External exposure reacts to obtain gel intermediate, and then gel intermediate is soaked in sodium alginate soln, instills calcium chloride solution,
Cross-linking reaction, closing are placed, and washing obtains gel carrier.
2. microbial bacterial agent according to claim 1, which is characterized in that the compound microorganism bacterium powder is by following system
What Preparation Method obtained: first by bacillus cereus ACCC03288, Trichoderma harzianum ACCC30371, Pseudomonas chlororaphis
ACCC19853, Paecilomyces lilacinus ACCC30639, Streptomyces hygrospinosus ACCC40018 bacterial strain individually carry out inoculation training
Feeding, flocculation, filters pressing, drying and crushing, obtain corresponding bacterium powder, then mix gained bacterium powder to get described compound micro-
Biological bacteria powder.
3. microbial bacterial agent according to claim 1, which is characterized in that the gel carrier is to be prepared by the following method
It obtains: acrylamide and sodium acrylate being first added into polyvinyl alcohol water solution, add crosslinking agent and photoinitiator mixing
Uniformly, ultraviolet irradiation reacts, and obtains gel intermediate, then gel intermediate is soaked in sodium alginate soln, instills chlorine
Change calcium solution, cross-linking reaction 30~40 minutes, closing was placed 10~12 hours, and washing obtains gel carrier.
4. microbial bacterial agent according to claim 3, which is characterized in that after crosslinking agent is added, letting nitrogen in and deoxidizing adds light
Initiator is uniformly mixed, wherein the specific method of letting nitrogen in and deoxidizing is: nitrogen is bubbled 10~30 minutes.
5. microbial bacterial agent according to claim 3, which is characterized in that polyvinyl alcohol water solution, acrylamide, acrylic acid
Sodium, crosslinking agent, photoinitiator, sodium alginate soln, calcium chloride solution mass ratio be 1:0.2~0.3:1.5~1.8:0.1~
0.2:0.03~0.04:1~2:6~8, polyvinyl alcohol water solution, sodium alginate soln, calcium chloride solution mass concentration successively
It is 5~8%, 2~3%, 2~3%.
6. according to the described in any item microbial bacterial agents of claim 3-6, which is characterized in that the process conditions of ultraviolet irradiation reaction
Are as follows: ultraviolet light irradiation reaction 5~7 hours of wavelength 365nm, power 400W.
7. a kind of preparation method for the microbial bacterial agent for preventing and treating ring rot of apple, feature described in any one of claim 1~6
It is, first microbial bacterial agent is added to the water and stirs evenly to obtain aqueous solution, then pours into gel carrier in aqueous solution, with 10~
15L/ minutes air velocity blowing airs, 1000~1500 revs/min are stirred 30~40 minutes, and closing stands 5~8 hours, are done
It is dry to get the microbial bacterial agent.
8. application of the microbial bacterial agent described in any one of claim 1~6 in prevention and treatment ring rot of apple.
9. 100 times of weight are added in microbial bacterial agent by the application method of microbial bacterial agent described in any one of claim 1~6
Water in dilute dissolution, be then applied to trunk rough bark surface, applying amount is 0.3~0.5mL/cm2。
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