CN109266577A - A kind of hybrid bacterial strain fermentation liquid and its preparation and application - Google Patents
A kind of hybrid bacterial strain fermentation liquid and its preparation and application Download PDFInfo
- Publication number
- CN109266577A CN109266577A CN201811100346.3A CN201811100346A CN109266577A CN 109266577 A CN109266577 A CN 109266577A CN 201811100346 A CN201811100346 A CN 201811100346A CN 109266577 A CN109266577 A CN 109266577A
- Authority
- CN
- China
- Prior art keywords
- fermentation broth
- bacterial strain
- strain fermentation
- strain
- mixed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 84
- 230000004151 fermentation Effects 0.000 title claims abstract description 84
- 239000007788 liquid Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 title abstract description 50
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 33
- 241000894006 Bacteria Species 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 239000008223 sterile water Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 241001149558 Trichoderma virens Species 0.000 claims 4
- 238000001471 micro-filtration Methods 0.000 claims 2
- 239000011148 porous material Substances 0.000 claims 2
- 239000011550 stock solution Substances 0.000 claims 2
- 241000219998 Philenoptera violacea Species 0.000 claims 1
- 241000499912 Trichoderma reesei Species 0.000 claims 1
- 240000006482 Trillium sessile Species 0.000 claims 1
- 239000000243 solution Substances 0.000 claims 1
- 241000223259 Trichoderma Species 0.000 abstract description 30
- 241000223600 Alternaria Species 0.000 abstract description 10
- 230000002265 prevention Effects 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 3
- 239000000575 pesticide Substances 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 17
- 241000412366 Alternaria mali Species 0.000 description 9
- 230000001408 fungistatic effect Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 8
- 238000011534 incubation Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 230000008485 antagonism Effects 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 230000003385 bacteriostatic effect Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000726221 Gemma Species 0.000 description 3
- 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 3
- 235000002595 Solanum tuberosum Nutrition 0.000 description 3
- 244000061456 Solanum tuberosum Species 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000010603 pastilles Nutrition 0.000 description 3
- 230000002688 persistence Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241001361634 Rhizoctonia Species 0.000 description 2
- 241001558929 Sclerotium <basidiomycota> Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000443 biocontrol Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 235000012015 potatoes Nutrition 0.000 description 2
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 1
- 239000005760 Difenoconazole Substances 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000005867 Iprodione Substances 0.000 description 1
- 239000005800 Kresoxim-methyl Substances 0.000 description 1
- 239000005802 Mancozeb Substances 0.000 description 1
- 229930182764 Polyoxin Natural products 0.000 description 1
- 241000233639 Pythium Species 0.000 description 1
- 241000223262 Trichoderma longibrachiatum Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 235000015895 biscuits Nutrition 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
- 230000007812 deficiency Effects 0.000 description 1
- BQYJATMQXGBDHF-UHFFFAOYSA-N difenoconazole Chemical compound O1C(C)COC1(C=1C(=CC(OC=2C=CC(Cl)=CC=2)=CC=1)Cl)CN1N=CN=C1 BQYJATMQXGBDHF-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- ONUFESLQCSAYKA-UHFFFAOYSA-N iprodione Chemical compound O=C1N(C(=O)NC(C)C)CC(=O)N1C1=CC(Cl)=CC(Cl)=C1 ONUFESLQCSAYKA-UHFFFAOYSA-N 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- ZOTBXTZVPHCKPN-HTXNQAPBSA-N kresoxim-methyl Chemical compound CO\N=C(\C(=O)OC)C1=CC=CC=C1COC1=CC=CC=C1C ZOTBXTZVPHCKPN-HTXNQAPBSA-N 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- YEBIHIICWDDQOL-YBHNRIQQSA-N polyoxin Polymers O[C@@H]1[C@H](O)[C@@H](C(C=O)N)O[C@H]1N1C(=O)NC(=O)C(C(O)=O)=C1 YEBIHIICWDDQOL-YBHNRIQQSA-N 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to biological pesticide technical fields, and in particular to a kind of hybrid bacterial strain fermentation liquid and its preparation and application.By preparing a kind of hybrid bacterial strain fermentation liquid, which is to be mixed with to obtain by the ratio that bacillus amyloliquefaciens fermentation liquid and long shoot trichoderma T6 bacterial strain fermentation liquor are 1:1-3 by volume, has effectively acted as the effect of prevention and treatment alternaria leaf spot of apple.
Description
Technical field
The invention belongs to biological pesticide technical fields, and in particular to a kind of hybrid bacterial strain fermentation liquid and its preparation and application.
Background technique
Alternaria leaf spot of apple main harm Apple Leaves, cause Apple Leaves early fallout, and tree vigo(u)r is weak.The disease is at me
State's apple producing region generally occurs, and seriously affects apple production and quality, has become an important factor for restricting apple industry.
Currently, alternaria leaf spot of apple is based on Agro-chemicals control, common medicament include polyoxin, difenoconazole, iprodione,
Mancozeb, carbendazim and kresoxim-methyl etc., but Agro-chemicals control be easy to cause environmental pollution, pesticide residue and drug resistance etc.
A series of problems, and biological control is increasingly subject to the attention of domestic and foreign scholars because it has the characteristics that high-efficiency low-toxicity.Currently, state
Inside and outside related utilization compound bacteria collaboration controlling plant diseases have relevant report.It is reported that Trichoderma is true to various plants cause of disease
Bacterium has preferable antagonism, such as Rhizoctonia (Rhizoctonia spp.), Fusarium (Fusarium spp.), corruption
Mould category (Pythium spp.) and sclerotium (Sclerotium spp.) etc..Meanwhile bacillus amyloliquefaciens are widely present
In nature, because it has many advantages, such as that the speed of growth is fast, secondary metabolite is abundant, utility value is high and to safety of human and livestock,
It has been used as one kind to have very much potential biocontrol microorganisms resource at present to be widely used in industry, medicine and agricultural production, but
That there are prevention and treatment ranges is narrow for prevention and treatment some researches show that single microbial strains to plant disease, effect is low and control efficiency is unstable
It is fixed, and to the drawbacks such as the adaptability and resistance of environment is poor.Therefore, it develops hybrid bacterial strain fermentation liquid and effectively controls phytopathy
Evil has become current research hotspot both domestic and external, but the complex microorganism preparations about prevention and treatment alternaria leaf spot of apple both at home and abroad
Research have not been reported.
Summary of the invention
Regarding to the issue above and the deficiencies in the prior art, the present invention provides a kind of hybrid bacterial strain fermentation liquid, the Mixed Microbes
Strain fermentation liquid is mixed by the ratio that bacillus amyloliquefaciens fermentation liquid and long shoot trichoderma T6 bacterial strain fermentation liquor are 1:1-3 by volume
Conjunction is prepared.
Further, the hybrid bacterial strain fermentation liquid is by bacillus amyloliquefaciens fermentation liquid and long shoot trichoderma T6 bacterial strain
Fermentation liquid is that the ratio of 1:3 is mixed with to obtain by volume.
The present invention also provides a kind of preparation methods of above-mentioned hybrid bacterial strain fermentation liquid, method includes the following steps:
1. the preparation of long shoot trichoderma T6 bacterial strain fermentation liquor: the long shoot trichoderma T6 bacterial strain bacterium of activation culture 6d on PDA plate
Middle addition 5-6mL sterile water is fallen, using sterile small its conidium of writing brush swipe, and it is made sufficiently to fall off in sterile water,
Obtain the stoste of conidial suspension.Then, using blood counting chamber count original liquid concentration, and make its concentration 1.0 ×
107A/mL places 4 DEG C of refrigerators and saves backup；
It takes above-mentioned spore suspension 1mL to be added in sterilized 100mL PDB culture solution, is placed in 30 DEG C of shaking tables,
180rpm/min is 4 DEG C through temperature after cultivating 5d, and the centrifuge that rate is 8000rpm/min is centrifuged 20min, and through the double-deck nothing
Bacterium filter paper filters 2 times.Then, filtered filtrate filters 2 removing mycelia by the nuclepore membrane filter in 0.22 μm of aperture
And spore, without fermented liquid is obtained, it is spare to place 4 DEG C of refrigerators；
2. the preparation of bacillus amyloliquefaciens fermentation liquid: taking the activated solution starch gemma of a ring with the collarium that connects sterilized
The single colonie of bacillus is put into sterilized NB culture solution, is placed in 30 DEG C of shaking tables, and 180rpm/min is through temperature after cultivating 3d
4 DEG C, the centrifuge that rate is 8000rpm/min is centrifuged 20min, and filters 2 times through double-layer sterile filter paper.Then, filtered
Filtrate is filtered 2 times by the nuclepore membrane filter in 0.22 μm of aperture, and it is spare to place 4 DEG C of refrigerators for supernatant after filtering；
3. the preparation of hybrid bacterial strain fermentation liquid: 1. 2. bacillus amyloliquefaciens fermentation liquid that above-mentioned steps are prepared with step
It is mixed with to obtain hybrid bacterial strain fermentation liquid with ratio that long shoot trichoderma T6 bacterial strain fermentation liquor is 1:1-3 by volume.
Further, the step of the method 3. in bacillus amyloliquefaciens fermentation liquid and long shoot trichoderma T6 bacterial strain fermentation liquor
Volume ratio be 1:3.
In addition the answering in prevention and treatment alternaria leaf spot of apple the present invention also provides a kind of above-mentioned hybrid bacterial strain fermentation liquid
With.
In addition the present invention also provides the mixing that the preparation method by a kind of above-mentioned hybrid bacterial strain fermentation liquid is prepared
Application of the bacterial strain fermentation liquor in prevention and treatment alternaria leaf spot of apple.
The beneficial effects of the present invention are:
1. overcoming the drawbacks such as high residue existing for chemical classes fungicide and environmental pollution, to green and have to Apple Industry
Machine direction, which is developed, to have great importance.
2. being provided with exploitation alternaria leaf spot of apple microbial control resource using foundation to excavate, and to improve single life
Preventing microorganism bacterial strain provides the control efficiency of alternaria leaf spot of apple new thinking and strategy；
3. providing possible ways to research and develop novel, efficient disease-resistant compound microbial preparation；
4. providing new method for effectively preventing for alternaria leaf spot of apple, economy and society effect is increased income for apple industry
Benefit.
Detailed description of the invention
Fig. 1: long shoot trichoderma T6 bacterial strain and bacillus amyloliquefaciens compatibility measurement chart；
Fig. 2: various concentration long shoot trichoderma T6 bacterial strain fermentation liquor fungistatic effect figure；
Fig. 3: various concentration bacillus amyloliquefaciens fermentation liquid fungistatic effect figure；
Fig. 4: hybrid bacterial strain fermentation liquid fungistatic effect figure；
Note: A, B, C in Fig. 1 are respectively as follows: the compatibility in the PDA culture medium, compatibility on NA culture medium, stereoscopic micro-
Observation situation under mirror；A, B, C, D in Fig. 2 are respectively as follows: control, bacillus amyloliquefaciens and long shoot trichoderma T6 bacterial strain fermentation liquor
Concentration is 1%, 3%, 4%；A, B, C, D in Fig. 3 are respectively as follows: control, bacillus amyloliquefaciens and long shoot trichoderma T6 bacterial strain and send out
Zymotic fluid concentration is 1%, 3%, 4%；A, B, C, D in Fig. 4 are respectively as follows: control, bacillus amyloliquefaciens and long shoot trichoderma T6 bacterium
The volume ratio of strain fermentation liquid is 1:1,1:2,1:3.
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 invention, rather than the whole invented.Based on the embodiments of the present invention, ordinary skill people
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
Experimental material used in the following example and instrument include:
For trying biomaterial: long shoot trichoderma (Trichoderma longibrachiatum) T6 bacterial strain, solution starch gemma bar
Bacterium (Bacillus amyloliquefaciens) and Alternaria mali (Alternaria mali) are by agriculture of gansu
University plant protection institute plant aetiology laboratory provides.
For trying culture medium: potato glucose sugar agar (PDA) culture medium (200g peeled potatoes, 20g glucose sugar, 10g fine jade
Rouge, distilled water are settled to 1000mL)；NA culture medium (3g beef extract, 10g peptone, 5g NaCl, 10g agar, distilled water constant volume
To 1000mL)；PDB culture medium (200g peeled potatoes, 20g glucose sugar, distilled water are settled to 1000mL)；NB culture medium (3g
Beef extract, 10g peptone, distilled water are settled to 1000mL).
The preparation of a kind of hybrid bacterial strain fermentation liquid of embodiment 1 and its to the inhibiting effect of Alternaria mali
One, process is tested:
1. the preparation of long shoot trichoderma T6 bacterial strain spore suspension and fermentation liquid: the long shoot of activation culture 6d on PDA plate
5-6mL sterile water is added in trichoderma T6 bacterial strain bacterium colony, using sterile small its conidium of writing brush swipe, and it is made sufficiently to fall off
The stoste of conidial suspension is arrived in sterile water.Then, original liquid concentration is counted using blood counting chamber, and makes it
Concentration is 1.0 × 107A/mL places 4 DEG C of refrigerators and saves backup；
It takes above-mentioned spore suspension 1mL to be added in sterilized 100mL PDB culture solution, is placed in 30 DEG C of shaking tables,
180rpm/min is 4 DEG C through temperature after cultivating 5d, and the centrifuge that rate is 8000rpm/min is centrifuged 20min, and through the double-deck nothing
Bacterium filter paper filters 2 times.Then, filtered filtrate filters 2 removing mycelia by the nuclepore membrane filter in 0.22 μm of aperture
And spore, without fermented liquid is obtained, it is spare to place 4 DEG C of refrigerators；
2. the preparation of bacillus amyloliquefaciens fermentation liquid: taking the activated solution starch gemma of a ring with the collarium that connects sterilized
The single colonie of bacillus is put into sterilized NB culture solution, is placed in 30 DEG C of shaking tables, and 180rpm/min is through temperature after cultivating 3d
4 DEG C, the centrifuge that rate is 8000rpm/min is centrifuged 20min, and filters 2 times through double-layer sterile filter paper.Then, filtered
Filtrate is filtered 2 times by the nuclepore membrane filter in 0.22 μm of aperture, and it is spare to place 4 DEG C of refrigerators for supernatant after filtering；
3. the preparation of hybrid bacterial strain fermentation liquid: 1. 2. bacillus amyloliquefaciens fermentation liquid that above-mentioned steps are prepared with step
It is mixed with to obtain hybrid bacterial strain fermentation liquid with ratio that long shoot trichoderma T6 bacterial strain fermentation liquor is 1:1,1:2,1:3 by volume.
4. the compatibility of bacillus amyloliquefaciens and long shoot trichoderma T6 bacterial strain measures: by bacillus amyloliquefaciens, long shoot wood
After mould T6 bacterial strain activates on solid medium, by two kinds of strain inoculateds to same culture medium in a manner of opposite culture, with list
One bacterial strain is control, and 28 DEG C of culture 3d measure colony diameter, whether affinity observe two kinds of biocontrol microorganisms.
5. fermentation liquid Antibacterial Activity
5.1 single fermentation liquid Antibacterial Activities: each 1mL of above two fermentation liquid is taken to be added separately to 70mL with liquid-transfering gun
In the sterilized PDA culture medium for being cooled to 45 DEG C, pours into and pastille culture medium is made in sterilizes culture dish.Then, it is beaten using sterile
Hole device produces the bacteria cake of diameter 5mm in the Alternaria mali colony edge of 28 DEG C of constant temperature incubation 5d, and by pure culture biscuits involvng inoculation
To drug containing PDA plate, it is placed in 28 DEG C of constant temperature incubations.Test is to add the PDA culture medium of 1mL sterile water as control, each processing
It is repeated 3 times with control, and starts to measure colony diameter after cultivating 3d, calculate bacteriostasis rate.
Bacteriostasis rate (%)=[(control colony diameter-processing colony diameter)/(control colony diameter -0.5)], 0.5 in formula
For the diameter (cm) for accessing bacteria cake.
5.2 hybrid bacterial strain fermentation liquid Antibacterial Activities: with liquid-transfering gun by above-mentioned three kinds of different proportions (1:1,1:2,1:3)
The total 1mL of mixed fermentation liquid be added in the sterilized PDA culture medium of 70mL, pastille culture medium is made, is repeated 3 times.Using
The punch of sterilizing produces the bacteria cake that diameter is 5mm in the Alternaria mali colony edge of 28 DEG C of constant temperature incubation 5d, and
Access constant temperature incubation in pastille culture medium.Test is each to handle and compare counterpoise using single fermentation liquid and sterile water as control
It is 3 times multiple, and start to measure colony diameter after cultivating 3d, calculate bacteriostasis rate.
Bacteriostasis rate (%)=[(control colony diameter-processing colony diameter)/(control colony diameter -0.5)], 0.5 in formula
For the diameter (cm) for accessing bacteria cake.
Two, test results and analysis
1. the compatibility testing result of long shoot trichoderma T6 bacterial strain and bacillus amyloliquefaciens
The long shoot trichoderma T6 bacterial strain activated and bacillus amyloliquefaciens are inoculated in the PDA of same blank, NA training respectively
It supports and carries out colony diameter measurement after cultivating 3-7d on base.The result shows that two bacterium all make without generating antagonism on two kinds of culture mediums
With.As shown in Figure 1, it can be observed that the spore of long shoot trichoderma T6 bacterial strain can almost surround solution starch bud under stereomicroscope
Spore bacillus, and compared with the control without mutual antagonism, it was demonstrated that two bacterium have good compatibility.
2. the bacteriostatic activity testing result of long shoot trichoderma T6 bacterial strain fermentation liquor
Alternaria mali is cultivated in the long shoot trichoderma T6 bacterial strain fermentation liquor PDA culture medium containing different volumes
Colony diameter measurement is carried out after 3-7d, table 1 is the results show that with its bacteriostasis rate of the increase of long shoot trichoderma T6 bacterial strain fermentation liquor volume
Also correspondingly increasing, fungistatic effect is best when volume ratio is 4%, and 7d bacteriostasis rate is up to 81.80% (p < 0.05), and 3% time
It, 7d is worst up to 71.55% (p < 0.05), 1%, and 7d is 70.55% (p < 0.05), is obtained by data, three kinds of concentration
With the extension of incubation time, bacteriostasis rate is all increasing.Fig. 2 shows that long shoot trichoderma T6 bacterial strain fermentation liquor falls leaves to apple spot
The antagonism of germ has persistence.
The fungistatic effect of 1 long shoot trichoderma T6 bacterial strain fermentation liquor of table
3. the bacteriostatic activity testing result of bacillus amyloliquefaciens fermentation liquid
Alternaria mali is cultivated in the bacillus amyloliquefaciens fermentation liquid PDA culture medium containing different volumes
After 3-7d carry out colony diameter measurement, table 2 the results show that with bacillus amyloliquefaciens fermentating liquid volume increase, it is antibacterial
Rate also increases accordingly, and fungistatic effect is best when concentration is 4%, and 7d bacteriostasis rate takes second place up to 75.82% (p < 0.05), 3%, the
For 7d up to 64.44% (p < 0.05), 1% is worst, and 7d is obtained up to 61.59% (p < 0.05) by data, three kinds of concentration with
The extension of incubation time, bacteriostasis rate are also increasing.Fig. 3 shows bacillus amyloliquefaciens fermentation liquid to alternaria leaf spot of apple
The inhibiting effect of opportunistic pathogen has good persistence.
The fungistatic effect of 2 bacillus amyloliquefaciens fermentation liquid of table
4. the bacteriostatic activity testing result of hybrid bacterial strain fermentation liquid
Alternaria mali is being mixed into hair with bacillus amyloliquefaciens different proportion containing long shoot trichoderma T6 bacterial strain
In the PDA culture medium of zymotic fluid cultivate 3-7d after, table 3 the results show that with long shoot trichoderma T6 strain fermentation liquid proportional increase,
Bacteriostasis rate also correspondingly increases, especially its ratio be fungistatic effect when 1:3 is best, 7d bacteriostasis rate up to 93.88% (p < 0.05),
1:2 takes second place, and for 7d up to 86.49% (p < 0.05), 1:1 is worst, and 7d bacteriostasis rate is 86.21% (p < 0.05), and data are shown,
Fermentation liquid is mixed in three kinds of ratios, and with the extension of incubation time, bacteriostasis rate is also increasing.Fig. 4 shows two bacterium mixed fermentations
Liquid has better inhibiting effect to Alternaria mali, and its inhibiting effect has preferable persistence.
The fungistatic effect of table 3 long shoot trichoderma T6 bacterial strain and bacillus amyloliquefaciens mixed fermentation liquid
To sum up, this test is using long shoot trichoderma T6 bacterial strain and bacillus amyloliquefaciens as material, using bacterium colony growth rate method
Control efficiency when its fermentation liquid is used alone and is used in mixed way is compared, the two mixed fermentation liquid is shown and apple spot is fallen
The control efficiency of leaf disease is significantly higher than any one fermentation liquid of exclusive use, no matter and its fermentation liquid be single use or
Being used in mixed way all has lasting bacteriostatic activity.
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811100346.3A CN109266577A (en) | 2018-09-20 | 2018-09-20 | A kind of hybrid bacterial strain fermentation liquid and its preparation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811100346.3A CN109266577A (en) | 2018-09-20 | 2018-09-20 | A kind of hybrid bacterial strain fermentation liquid and its preparation and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109266577A true CN109266577A (en) | 2019-01-25 |
Family
ID=65197153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811100346.3A Pending CN109266577A (en) | 2018-09-20 | 2018-09-20 | A kind of hybrid bacterial strain fermentation liquid and its preparation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109266577A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150223472A1 (en) * | 2001-02-02 | 2015-08-13 | Novozymes Bioag A/S | Increasing plant yield with bacterial/fungal combinations |
| CN105368739A (en) * | 2015-11-06 | 2016-03-02 | 山东省烟台市农业科学研究院 | Bacillus amyloliquefaciens strain capable of resisting fruit tree pathogenic fungi in broad spectrum and application thereof |
| CN106676047A (en) * | 2017-02-23 | 2017-05-17 | 邢台学院 | Bacillus amyloliquefaciens HM-6, and preparation method and application of microbial inoculum thereof |
-
2018
- 2018-09-20 CN CN201811100346.3A patent/CN109266577A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150223472A1 (en) * | 2001-02-02 | 2015-08-13 | Novozymes Bioag A/S | Increasing plant yield with bacterial/fungal combinations |
| CN105368739A (en) * | 2015-11-06 | 2016-03-02 | 山东省烟台市农业科学研究院 | Bacillus amyloliquefaciens strain capable of resisting fruit tree pathogenic fungi in broad spectrum and application thereof |
| CN106676047A (en) * | 2017-02-23 | 2017-05-17 | 邢台学院 | Bacillus amyloliquefaciens HM-6, and preparation method and application of microbial inoculum thereof |
Non-Patent Citations (5)
| Title |
|---|
| 代志: "兼具菌肥功能生防菌分离鉴定及其生物活性评价", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 张建华等: "长枝木霉T6发酵液稳定性及粗提物抑菌活性", 《农药》 * |
| 牛鑫斌等: "长枝木霉T6水分散粒剂对3种植物病害的生防效果", 《甘肃农业大学学报》 * |
| 赵云福等: "解淀粉芽孢杆菌与九种农药的相容性及对苹果斑点落叶病田间防效的评价", 《北方园艺》 * |
| 陈哲等: "解淀粉芽孢杆菌抑菌机制的研究进展", 《生物技术通报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101423812B (en) | Bacillus amyloliquefaciens and microbial preparation and preparation method thereof | |
| CN102676398B (en) | Ginkgo biloba endophytes | |
| CN105368747B (en) | A strain of Bacillus amyloliquefaciens and its application | |
| US11033032B2 (en) | Leclercia adecarboxylata biocontrol strain efficiently inhibiting production of aflatoxins by Aspergillus flavus and application thereof | |
| CN104342389A (en) | Streptomyces strains and joint application thereof in prevention and treatment of cucumber fusarium wilt | |
| CN103243030A (en) | Lecanicilliumpsalliotae strain used for preventing and treating diaphorina citri | |
| CN107446831A (en) | A kind of bio-control yeast GA8 and its application | |
| CN103484500B (en) | Bacterial CD-126 fermentation solution and application thereof | |
| CN109868236A (en) | One plant of Jie meter La series bacillus, its tunning and preparation method and application | |
| CN112266879A (en) | Trichoderma harzianum SQ-18 strain and application thereof in preventing and treating turfgrass diseases | |
| CN102719364B (en) | Trichoderma harzianum strain and application in prevention and control of phytophthora capsici Leonian thereof | |
| CN105062897B (en) | The Trichoderma viride of one plant height production chlamydospore and its application | |
| CN102061269A (en) | Culture method for improving control efficacy of marine yeasts on fruit diseases and culture medium used in method | |
| CN113604376B (en) | Sugarcane endophytic bacillus subtilis and application thereof | |
| CN103352020A (en) | Method for preparing microbial preparation of bacillus sp and application thereof | |
| CN103773699B (en) | The application of one strain penicillium purpurogenum and biotechnological formulation and control peronophythora litchi | |
| CN109266577A (en) | A kind of hybrid bacterial strain fermentation liquid and its preparation and application | |
| Li et al. | Antagonistic mechanism of Fusarium oxysporum of soybean root rot by Bacillus subtilis | |
| CN107937479B (en) | A method of measurement biocontrol bacteria metabolite antagonistic activity | |
| CN103289931B (en) | Bacillus vallismortis strain SJ and application thereof in preparation of tobacco antiviral preparation and promoter | |
| CN108060088B (en) | Culture medium for magnaporthe grisea culture and spore production and preparation method thereof | |
| CN107058183B (en) | Bacillus methylotrophicus, and biocontrol microbial inoculum and application thereof | |
| CN105733984B (en) | Bacillus subtilis and its application in terms of control of leaf spot of corn | |
| CN103255065A (en) | Method for preparing anti-phytopathogen fermented liquid for camptotheca acuminate endophytic fungi | |
| CN103087949B (en) | Biocontrol endophytic actinomycetes- streptomyces sioyaensis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190125 |