CN105494431A - Lipopeptide and nano-titania compound preparation and preparation method and application thereof - Google Patents
Lipopeptide and nano-titania compound preparation and preparation method and application thereof Download PDFInfo
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- CN105494431A CN105494431A CN201510991377.2A CN201510991377A CN105494431A CN 105494431 A CN105494431 A CN 105494431A CN 201510991377 A CN201510991377 A CN 201510991377A CN 105494431 A CN105494431 A CN 105494431A
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- lipopeptid
- nano titanium
- titanium oxide
- fermentation
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- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 43
- 108010028921 Lipopeptides Proteins 0.000 title abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 39
- 230000001580 bacterial effect Effects 0.000 claims abstract description 24
- 230000001737 promoting effect Effects 0.000 claims abstract description 5
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 claims description 92
- 239000000203 mixture Substances 0.000 claims description 81
- -1 titanium oxide compound Chemical class 0.000 claims description 62
- 238000009472 formulation Methods 0.000 claims description 59
- 238000000855 fermentation Methods 0.000 claims description 56
- 230000004151 fermentation Effects 0.000 claims description 56
- 239000007788 liquid Substances 0.000 claims description 50
- 239000000843 powder Substances 0.000 claims description 46
- 240000003768 Solanum lycopersicum Species 0.000 claims description 37
- 238000001556 precipitation Methods 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 26
- 150000003839 salts Chemical class 0.000 claims description 24
- 241000196324 Embryophyta Species 0.000 claims description 23
- 238000003756 stirring Methods 0.000 claims description 20
- 235000013619 trace mineral Nutrition 0.000 claims description 20
- 239000011573 trace mineral Substances 0.000 claims description 20
- 239000010936 titanium Substances 0.000 claims description 19
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical compound [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 claims description 17
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 14
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 230000012010 growth Effects 0.000 claims description 10
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 10
- 239000012266 salt solution Substances 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 9
- DCKVFVYPWDKYDN-UHFFFAOYSA-L oxygen(2-);titanium(4+);sulfate Chemical compound [O-2].[Ti+4].[O-]S([O-])(=O)=O DCKVFVYPWDKYDN-UHFFFAOYSA-L 0.000 claims description 9
- 230000001105 regulatory effect Effects 0.000 claims description 9
- 229910000348 titanium sulfate Inorganic materials 0.000 claims description 9
- LLZRNZOLAXHGLL-UHFFFAOYSA-J titanic acid Chemical compound O[Ti](O)(O)O LLZRNZOLAXHGLL-UHFFFAOYSA-J 0.000 claims description 8
- 244000068988 Glycine max Species 0.000 claims description 7
- 235000010469 Glycine max Nutrition 0.000 claims description 7
- 229910021529 ammonia Inorganic materials 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 229960005190 phenylalanine Drugs 0.000 claims description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 abstract description 33
- 230000000694 effects Effects 0.000 abstract description 5
- 230000008635 plant growth Effects 0.000 abstract description 4
- 230000002265 prevention Effects 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 3
- 241000227653 Lycopersicon Species 0.000 abstract 4
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 230000008569 process Effects 0.000 description 23
- 239000007921 spray Substances 0.000 description 14
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 244000052769 pathogen Species 0.000 description 11
- 230000001717 pathogenic effect Effects 0.000 description 11
- 229910052719 titanium Inorganic materials 0.000 description 11
- 201000010099 disease Diseases 0.000 description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 230000001276 controlling effect Effects 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 241000589771 Ralstonia solanacearum Species 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 238000002441 X-ray diffraction Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000001354 calcination Methods 0.000 description 3
- 244000144992 flock Species 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000001935 peptisation Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002390 rotary evaporation Methods 0.000 description 3
- 238000005204 segregation Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 229910021653 sulphate ion Inorganic materials 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 229910003077 Ti−O Inorganic materials 0.000 description 2
- 229910003088 Ti−O−Ti Inorganic materials 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000003876 biosurfactant Substances 0.000 description 2
- YYRMJZQKEFZXMX-UHFFFAOYSA-N calcium;phosphoric acid Chemical compound [Ca+2].OP(O)(O)=O.OP(O)(O)=O YYRMJZQKEFZXMX-UHFFFAOYSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000002426 superphosphate Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000004408 titanium dioxide Substances 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 241000813090 Rhizoctonia solani Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical class OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000002154 agricultural waste Substances 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002389 environmental scanning electron microscopy Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007665 sagging Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
Classifications
-
- 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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
-
- 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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
- A01N59/20—Copper
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a lipopeptide and nano-titania compound preparation. The lipopeptide and nano-titania compound preparation is characterized by being prepared from, by mass, 0.075%-0.15% of lipopeptide, 0.2%-0.3% of nano-titania, 0.0069%-0.0092% of Zn<2+>, 0.005%-0.007% of Fe<2+>, 0.0052%-0.0091% of Cu<2>, 0.005%-0.0083% of Mn<2+> and the balance water. The invention further discloses a preparation method of the lipopeptide and nano-titania compound preparation and application of the lipopeptide and nano-titania compound preparation in preventing and treating tomato bacterial wilt and promoting tomato plant growth. According to the prepared lipopeptide and nano-titania compound preparation, after nano-titania and lipopeptide are combined, the stability of lipopeptide can be improved, the action time of lipopeptide can be prolonged, a good prevention and treatment effect on tomato bacterial wilt can be achieved, and tomato plant growth can be promoted.
Description
Technical field
The invention belongs to microbial pesticide preparation field, relate to a kind of lipopeptid-nano titanium oxide compound formulation and its preparation method and application.
Background technology
Bacterial wilt of tomato is the bacillary soil-borne disease that a kind of whole world caused by Ralstonia solanacearum (Ralstoniasolanacearum) extensively distributes.Their early stage, the petiole of plant lower blade occurs sagging, and rear blade is withered gradually, and adventive root appears in stem simultaneously, and plant is wilted and withered gradually fast.Light then the underproduction 20% ~ 40%, reaches more than 60% and even has no harvest time serious.There is larger genetic variation in Ralstonia solanacearum, there is no effective chemical prevention and control method at present.Effect is very micro-due to the complexity of bacterial wilt disease system for conventional prophylactico-therapeutic measures such as resistive breeding, field removing, shift of crops, integrated management etc.
Bacillus (Bacillusspp.) has complicated physiological characteristics diversity and substrate diversity, can produce high patience gemma and various antibacterial substance as polypeptide class and lipopeptid class and bacteriocin class antibiotic.Fat peptid-based surfactant is as a kind of biosurfactant (biosurfactant), permeability of cell membrane can be changed, there is the activity of the antimycotic of wide spectrum and suppression part bacterium, research shows, some fat peptide matters Rhizoctonia solani and Ralstonia solanacearum show good antagonistic activity, and it can be utilized to prevent and treat bacterial wilt, but its chemical instability, physics and enzyme lability, limit the development and application of fat peptid-based surfactant Related product.Meanwhile, higher production cost and lower output also limit the suitability for industrialized production of lipopeptid.
In recent years, composite is prepared in the interphase interaction of biomaterial and organic film, organic granular, inorganic clay material, sizable interest has been caused in academia, particularly the abundant novelty of inorganic nano submicron order material structure, good heat stability, surface-active are adjustable, cheap, effective compound of biomaterial and inorganic nano material, the structure of large biological molecule can be maintained very well, and likely can give biological substance new excellent specific property.Nano titanium oxide has the physics and chemistry characteristic of many excellences, and such as high-specific surface area, more surface atom and good stability, can be used as the carrier of protein, agricultural chemicals, insecticide, and can fixing metal ions.Nano titanium oxide can provide an applicable microenvironment with immobilized enzyme and other protein, maintains biologically active and the stability of enzyme and the protein be fixed.Relevant research shows, nano titanium oxide has certain inhibitory action for bacterial growth, and in addition, nano titanium oxide can also Promoting plant growth.
Trace element is required for plant, and research shows, foliage-spray liquid microelement can play some positive effects to the growth of plant and output.
Summary of the invention
The technical issues that need to address of the present invention are, provide a kind of lipopeptid-nano titanium oxide compound formulation.
The technical problem that the present invention also will solve is, provides the preparation method of above-mentioned lipopeptid-nano titanium oxide compound formulation.
The technical problem that the present invention also will solve is, provides the above-mentioned lipopeptid-application of nano titanium oxide compound formulation in control of plant bacterial wilt.
The technical problem that the present invention finally will solve is, provides above-mentioned lipopeptid-nano titanium oxide compound formulation promoting the application on tomato plant strain growth.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
A kind of lipopeptid-nano titanium oxide compound formulation, said preparation comprises the component of following mass percent:
Lipopeptid 0.075% ~ 0.15%;
Nano titanium oxide 0.2% ~ 0.3%;
Trace elements zn
2+0.0069% ~ 0.0092%, Fe
2+0.005% ~ 0.007%, Cu
2+0.0052% ~ 0.0091% and Mn
2+0.005% ~ 0.0083%;
All the other are water.
Wherein, described lipopeptid, is prepared as follows and obtains:
(1a) activate bacillus amyloliquefaciens, obtain seed liquor; The bacillus amyloliquefaciens of all energy fermenting and producing lipopeptids may be used to the lipopeptid in production the present invention, the Bacillus amyloliquefaciens strain XZ-173 of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation is preferably utilized in the present invention, preserving number is CGMCCNo.4160, and the specifying information of this bacterial strain has been open in the Chinese patent of 201010608579.1 at application number;
The method of concrete activation bacillus amyloliquefaciens is as follows: by the Bacillus amyloliquefaciens strain of freezing at the flat lining out of LB, be placed in activation in 30 DEG C of incubators, picking list colony inoculation in LB liquid nutrient medium, at 30 DEG C, cultivate 24h under the condition of 170rpm, obtain fermentation seed liquid;
(1b) be transferred in fermentation medium by seed liquor, regulate relative moisture in fermentation tank to be greater than 80%, lipopeptid is produced in fermentation;
(1c) after fermentation ends, by fermenting, the mixture obtained mixes according to according to 1g:10mL ratio with water, centrifugal, collects supernatant, supernatant pH is regulated to be 1 ~ 3 with hydrochloric acid, leave standstill, more centrifugal, collecting precipitation, precipitation uses carrene extracting, revolve and boil off except carrene, residue is by water-soluble solution and adjust pH to be 7.0, and last freeze drying obtains lipopeptid powder.
Wherein, described nano titanium oxide is prepared as follows and obtains:
(2a) to press the concentration of 0.1mol/L soluble in water for titanium sulfate, and add the ammonia spirit of 2 ~ 3mol/L, the pH making solution is 6 ~ 8, obtains mixture;
(2b) mixture is centrifugal, collecting precipitation, washes precipitation with water;
(2c) precipitation that step (2b) obtains is scattered in water, adds the aqueous hydrogen peroxide solution that volume ratio is 30%, make Ti in solution
4+and H
2o
2mol ratio be 1:3 ~ 5, stir, leave standstill 15 ~ 20 hours, obtain peroxo titanic acid sol;
(2d) peroxo titanic acid sol is dried formation xerogel at 60 ~ 75 DEG C, by xerogel grind into powder, calcine 1 ~ 2 hour under 400 ~ 600 DEG C of conditions, obtain nano titanium dioxide powder.
Wherein, described nano titanium oxide particle diameter is 10 ~ 40nm.
Titanium dioxide has that specific surface area is large, the skin effect of high adsorption capacity, good stability and other nanometer materials, surface has many activated centres, titanium dioxide can be utilized lipopeptid and trace element ion effectively to be combined, maintain the structure of lipopeptid well, improve the stability of trace element ion, and play the biological nature of lipopeptid better.
Wherein, described trace element is Zn
2+salt, Fe
2+salt, Cu
2+salt and Mn
2+any one or a few mixture in salt.
Described Zn
2+salt, Fe
2+salt, Cu
2+salt and Mn
2+salt can be the sulphate of above-mentioned ion, nitrate or hydrochloride etc., preferably sulfuric acid salt.
As preferably, described trace element is Zn
2+salt, Fe
2+salt, Cu
2+salt and Mn
2+the mixture of salt, described Zn
2+salt, Fe
2+salt, Cu
2+salt and Mn
2+the preferred ZnSO of mixture of salt
4, FeSO, CuSO
4and MnSO
4mixture, further preferably ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2the mixture of content difference 0.3 ~ 0.4g/L, 0.25 ~ 0.35g/L, 0.2 ~ 0.35g/L and the 0.15 ~ 0.25g/L of O, most preferably ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2the content of O is the mixture of 0.35g/L, 0.3g/L, 0.24g/L and 0.18g/L.
The preparation method of above-mentioned lipopeptid-nano titanium oxide compound formulation, the method comprises the steps:
(1) preparation of lipopeptid:
(1a) activate bacillus amyloliquefaciens, obtain seed liquor;
(1b) be transferred in fermentation medium by seed liquor, regulate relative moisture in fermentation tank to be greater than 80%, lipopeptid is produced in fermentation;
(1c), after fermentation ends, by fermenting, the mixture obtained mixes according to 1g:10mL ratio with water, centrifugal, collect supernatant, regulate supernatant pH to be 1 ~ 3 with hydrochloric acid, preferably regulate pH to leave standstill 12h at 2,4 DEG C, centrifugal again, collecting precipitation, precipitation uses carrene extracting, then revolves and boil off except carrene, residue is by water-soluble solution and adjust pH to be 7.0, and last freeze drying obtains lipopeptid powder;
(2) preparation of nano titanium oxide:
(2a) to press the concentration of 0.1mol/L soluble in water for titanium sulfate, adds the ammonia spirit of 2 ~ 3mol/L, make the pH of solution be 6 ~ 8, preferably regulates the pH of solution 7 to obtain mixture;
(2b) mixture is centrifugal, collecting precipitation, washes precipitation with water;
(2c) precipitation that step (2b) obtains is scattered in water, adds the aqueous hydrogen peroxide solution that volume ratio is 30%, make Ti in solution
4+and H
2o
2mol ratio be 1:3 ~ 5, Ti
4+and H
2o
2mol ratio be preferably 1:4, stir, leave standstill 15 ~ 20 hours, time of repose is preferably 15h, obtains peroxo titanic acid sol;
(2d) peroxo titanic acid sol is dried formation xerogel at 60 ~ 75 DEG C, preferably dry at 75 DEG C and form xerogel, by xerogel grind into powder, calcine 1 ~ 2 hour under 400 ~ 600 DEG C of conditions, preferably under 500 DEG C of conditions, calcine 1h, obtain nano titanium dioxide powder;
(3) preparation of liquid microelement: be dissolved in the water by trace element, obtains trace element water-soluble liquid;
(4) lipopeptid-nano titanium oxide compound formulation obtains: in the trace element water-soluble liquid that step (3) obtains, add lipopeptid that step (1) obtains and the nano titanium oxide that step (2) obtains successively, stirring shakes up, 20 ~ 40 DEG C, under 150 ~ 250rpm condition, vibration 10 ~ 30h, preferably 30 DEG C, 170rpm vibrates 24h, namely obtains lipopeptid-nano titanium oxide compound formulation.
In step (1b), described fermentation medium as follows equal proportion prepares: take 4.5 ~ 6kg analysis for soybean powder, 3 ~ 4.5kg stalk, 0.15 ~ 0.2kg starch and 0.15 ~ 0.2kg yeast extract, put into fermentation tank, add 1 ~ 2L inorganic salt solution, pH is regulated to be 7 ~ 8, the moisture content regulating medium is 40% ~ 60%, and described inorganic salt solution, its formula is: 1.0g/LKH
2pO
4, 0.5g/LMgSO
47H
2o, 0.5g/LKCl, 2 × 10
-3g/LL-phenyl alanine, 5 × 10
-3g/LMnSO
4, 0.16 × 10
-3g/LCuSO
4, 0.15 × 10
-3g/LFeSO
4.
In step (1b), fermentation process adopts temperature-variable fermentation, keep that throughput is 0.1 ~ 0.5vvm, tank pressure is 0.01 ~ 0.05Mpa during the fermentation, first stage fermentation temperature is 25 ~ 40 DEG C, fermentation 20 ~ 24h, the fermentation temperature of second stage is 28 ~ 40 DEG C, fermentation 16 ~ 24h, and ensures that the temperature of second stage fermentation is higher than first stage temperature 0 ~ 7 DEG C; Preferred elder generation at 30 DEG C of bottom fermentation 24h, then at 37 DEG C of bottom fermentation 20h.
In step (3), preferred scheme is by ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2o's is soluble in water by following concentration: 0.3 ~ 0.4g/LZnSO
47H
2o, 0.25 ~ 0.35g/LFeSO
47H
2o, 0.2 ~ 0.35g/LCuSO
45H
2o and 0.15 ~ 0.25g/LMnSO
4h
2o, most preferably by ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2o's is soluble in water by following concentration: 0.35g/LZnSO
47H
2o, 0.3g/LFeSO
47H
2o, 0.24g/LCuSO
45H
2o and 0.18g/LMnSO
4h
2o.
Above-mentioned lipopeptid-nano titanium oxide compound formulation being applied within protection scope of the present invention in control of plant bacterial wilt.
Above-mentioned lipopeptid-nano titanium oxide compound formulation is promoting being applied within protection scope of the present invention on tomato plant strain growth.
Beneficial effect:
1, detect through infrared spectrum, nano titanium oxide prepared by the present invention and lipopeptid and trace element ion effectively combine, not only well maintain the structure of lipopeptid, improve the stability of trace element ion, and play the biological nature of lipopeptid better.Test shows, lipopeptid provided by the invention-nano titanium oxide compound formulation to the control efficiency of bacterial wilt of tomato all apparently higher than being used alone trace element solution, lipopeptid-micro-complex liquid or nano titanium oxide-micro-complex liquid, illustrate that the combination of nano titanium oxide and lipopeptid in lipopeptid-nano titanium oxide compound formulation can improve the stability of lipopeptid better, extend the action time of lipopeptid, this lipopeptid-nano titanium oxide compound formulation can improve the control efficiency of bacterial wilt of tomato effectively.
2. lipopeptid-nano titanium oxide compound formulation of preparing of the present invention is through overtesting, can promote the growth of tomato overground part and underground part, have significant difference compared with control group, illustrate that this lipopeptid-nano titanium oxide compound formulation can promote tomato plant strain growth.
3. the present invention prepares one of lipopeptid-nano titanium oxide compound formulation raw material fat peptide matters and utilizes solid fermentation agricultural wastes to produce, the present invention adopts analysis for soybean powder and stalk mixture as primary solids fermentation substrate, not only effectively improve the output of fat peptide matters, save production cost, also reduce environmental pressure, improve the availability of resource.
4. the present invention produce that lipopeptid-nano titanium oxide compound formulation has environmental pollution is little, low toxicity, safety, production cost is low, and control and the effective advantage of growth-promoting, be applicable to the large area use of tomato planting region.
Accompanying drawing explanation
Fig. 1 nano titanium oxide X-ray diffraction (XRD) figure.
Fig. 2 nano titanium oxide transmission electron microscope (TEM) figure.
Fig. 3 nano titanium oxide ESEM (SEM) figure.
In Fig. 4 sample infrared spectrum (FT-IR) figure, figure, a represents nano titanium dioxide powder, and b represents the powder after nano titanium oxide-micro-complex liquid centrifugal drying, and c represents the powder after lipopeptid-nano titanium oxide compound formulation centrifugal drying.
Fig. 5 lipopeptid to the fungistatic effect figure of bacterial wilt of tomato pathogen (A.2000mg/L; B.1500mg/L; C.1000mg/L).
Fig. 6 greenhouse controlling experiment respectively processes disease index (DI) variation diagram, and * represents to there is significant difference, and NS represents to there is not significant difference (p<0.05).
Fig. 7 greenhouse controlling experiment respectively processes overground part dry weight, and different lowercase represents significant difference (p<0.05).
Fig. 8 greenhouse controlling experiment respectively processes underground part dry weight, and different lowercase represents significant difference (p<0.05).
Fig. 9 greenhouse growth-promoting tests each process overground part underground part dry weight and fresh weight of plant seedlings (A. overground part fresh weight; B. underground part fresh weight; C. overground part dry weight; D. underground part dry weight), different lowercase represents significant difference (p<0.05).
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Analysis for soybean powder and stalk, first dry 12h and remove most of moisture, grind away, pulverizing, cross 20 mesh sieves in 60 DEG C of baking ovens, the sample after sieving put into again baking mutually 60 DEG C dry 48h to constant weight, preserve stand-by.
Embodiment 1:
The preparation method of lipopeptid-nano titanium oxide compound formulation: (1) lipopeptid powder preparation: by Bacillus amyloliquefaciens strain XZ-173 (China Committee for Culture Collection of Microorganisms's common micro-organisms center of freezing, CGMCCNo.4160), line on LB flat board, be placed in activation in incubator, picking list colony inoculation is in LB liquid nutrient medium, at 30 DEG C, under the condition of 170rpm, cultivate 24h, obtain fermentation seed liquid; Take 4.5kg analysis for soybean powder, 3kg stalk, 0.15kg starch and 0.15kg yeast extract, put into 50L fermentation tank, add 1L inorganic salt solution, regulate substrate pH to be 7, ensure that moisture content is 40%, rotating speed is set to 50rpm stirring 60min and fully mixes matrix, sterilizing; Wherein, inorganic salt solution comprises (g/L): KH
2pO
41.0, MgSO
47H
2o0.5, KCl0.5, L-Phe 2 × 10
-3, MnSO
45 × 10
-3, CuSO
40.16 × 10
-3, FeSO
40.15 × 10
-3; Until temperature in fermentation tank cool and keep stable after, aseptically by 5% (v/w, mL/g) amount access seed liquor, stir, in regulating tank, relative moisture is greater than 80%, throughput 0.1vvm and tank pressure 0.01Mpa, front 24h temperature is set to 25 DEG C and ferments, and after 24h, temperature is set to 28 DEG C of fermentation 16h; Take the matrix after fermentation and water by 1:10 (w/v, g/mL) ratio mixing, shaken at room temperature 1h, centrifugal segregation insoluble matter, in supernatant, add HCl to pH is 1, occur white flock precipitate, stand at low temperature is spent the night, then with the centrifugal 20min collecting precipitation of 10000rpm, precipitation carrene carries out extracting, rotary evaporation removes organic solvent, and residue is by water-soluble solution and adjust pH to be neutral, and freeze drying obtains lipopeptid powder;
(2) nano titanium dioxide powder preparation: the concentration of titanium sulfate being pressed 0.1mol/L is soluble in water, the ammonia spirit adding 2mol/L generates white precipitate, makes the last pH of solution be 6, after solution centrifugal, and precipitation washes with water removes residual NH for several times
4 +and SO
4 2-, then white precipitate is scattered in water, adds the aqueous hydrogen peroxide solution (Ti of 30%
4+and H
2o
2mol ratio be 1:3) stir and make precipitation peptization, form yellow pertitanic acid (PTA) colloidal sol after stablizing 15 hours, PTA colloidal sol is dried at 60 DEG C and is formed xerogel, within 1 hour, obtains nano titanium dioxide powder after grinding with 400 DEG C of calcinings;
(3) liquid microelement preparation: by ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2o is soluble in water obtains liquid microelement, wherein ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2content 0.3g/L, 0.25g/L, 0.2g/L and the 0.15g/L respectively of O;
(4) lipopeptid-nano titanium oxide compound formulation obtains: in liquid microelement, add lipopeptid and nano titanium oxide successively, wherein, the content of lipopeptid is 0.075%, the content of nano titanium oxide is 0.2%, after stirring shakes up on shaking table with 20 DEG C, 150rpm vibrates 10h, namely obtains lipopeptid-nano titanium oxide compound formulation.
Embodiment 2:
The preparation method of lipopeptid-nano titanium oxide compound biological prevention and control agent: (1) lipopeptid powder preparation: bacillus amyloliquefaciens (Bacillusamyloliquefaciens) the strain X Z-173 of freezing is lined on LB flat board, be placed in activation in incubator, picking list colony inoculation is in LB liquid nutrient medium, at 30 DEG C, cultivate 24h under the condition of 170rpm, obtain fermentation seed liquid; Take 6kg analysis for soybean powder, 4.5kg stalk, 0.2kg starch and 0.2kg yeast extract, put into 50L fermentation tank, adds 2L inorganic salt solution, and regulate substrate pH to be 8, moisture content is 60%, rotating speed is set to 70rpm stirring 80min and fully mixes matrix, sterilizing; Wherein, inorganic salt solution comprises (g/L): KH
2pO
41.0, MgSO
47H
2o0.5, KCl0.5, L-Phe 2 × 10
-3, MnSO
45 × 10
-3, CuSO
40.16 × 10
-3, FeSO
40.15 × 10
-3; Until temperature in fermentation tank cool and keep stable after, aseptically by the amount access seed liquor of 25% (v/w, mL/g), stir, in regulating tank, relative moisture is greater than 80%, throughput 0.5vvm and tank pressure 0.05Mpa, and temperature is set to 40 DEG C of fermentation 48h; Take the matrix after fermentation and water by 1:10 (w/v, g/mL) ratio mixing, shaken at room temperature 2h, centrifugal segregation insoluble matter, in supernatant, add HCl to pH is 3, occur white flock precipitate, stand at low temperature is spent the night, then with the centrifugal 20min collecting precipitation of 10000rpm, precipitation carrene carries out extracting, rotary evaporation removes organic solvent, and residue is by water-soluble solution and adjust pH to be neutral, and freeze drying obtains lipopeptid powder;
(2) nano titanium dioxide powder preparation: the concentration of titanium sulfate being pressed 0.1mol/L is soluble in water, the ammonia spirit adding 3mol/L generates white precipitate, makes the last pH of solution be 8, after solution centrifugal, and precipitation washes with water removes residual NH for several times
4 +and SO
4 2-, then white precipitate is scattered in water, adds the aqueous hydrogen peroxide solution (Ti of 30%
4+and H
2o
2mol ratio be 1:5) stir and make precipitation peptization, form yellow pertitanic acid (PTA) colloidal sol after stablizing 20 hours, PTA colloidal sol is dried at 75 DEG C and is formed xerogel, within 2 hours, obtains nano titanium dioxide powder after grinding with 600 DEG C of calcinings;
(3) liquid microelement preparation: by ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2o is soluble in water obtains liquid microelement, wherein ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2content 0.4g/L, 0.35g/L, 0.35g/L and the 0.25g/L respectively of O;
(4) lipopeptid-nano titanium oxide compound formulation obtains: in liquid microelement, add lipopeptid and nano titanium oxide successively, wherein, the content of lipopeptid is 0.15%, the content of nano titanium oxide is 0.3%, after stirring shakes up on shaking table with 40 DEG C, 250rpm vibrates 30h, namely obtains lipopeptid-nano titanium oxide compound formulation.
Embodiment 3:
The preparation method of lipopeptid-nano titanium oxide compound biological prevention and control agent: (1) lipopeptid powder preparation: bacillus amyloliquefaciens (Bacillusamyloliquefaciens) the strain X Z-173 of freezing is lined on LB flat board, be placed in activation in incubator, picking list colony inoculation is in LB liquid nutrient medium, at 30 DEG C, 24h is cultivated, as fermentation seed liquid under the condition of 170rpm; Take 5.67kg analysis for soybean powder, 3.67kg stalk, 0.17kg starch and 0.18kg yeast extract, put into 50L fermentation tank, add 1L inorganic salt solution, regulate substrate pH to be 7.5, moisture content is 55%, rotating speed is set to 70rpm stirring 60min and fully mixes matrix, sterilizing; Wherein, inorganic salt solution comprises (g/L): KH
2pO
41.0, MgSO
47H
2o0.5, KCl0.5, L-Phe 2 × 10
-3, MnSO
45 × 10
-3, CuSO
40.16 × 10
-3, FeSO
40.15 × 10
-3; Until temperature in fermentation tank cool and keep stable after, aseptically by 10% (v/w, mL/g) amount access seed liquor, stir, in regulating tank, relative moisture is greater than 80%, throughput 0.3vvm and tank pressure 0.03Mpa, front 24h temperature is set to 30 DEG C and ferments, and after 24h, temperature is set to 37 DEG C of fermentation 20h; Take the matrix after fermentation and water by 1:10 (w/v, g/mL) ratio mixing, shaken at room temperature 1h, centrifugal segregation insoluble matter, in supernatant, add HCl to pH is 2.0, there is white flock precipitate, 4 DEG C of hold over night, then with the centrifugal 20min collecting precipitation of 10000rpm, precipitation carrene carries out extracting, rotary evaporation removes organic solvent, and residue is by water-soluble solution and adjust pH to be neutral, and freeze drying obtains lipopeptid powder;
(2) nano titanium dioxide powder preparation: the concentration of titanium sulfate being pressed 0.1mol/L is soluble in water, the ammonia spirit adding 3mol/L generates white precipitate, makes the last pH of solution be 7, after solution centrifugal, and precipitation washes with water removes residual NH for several times
4 +and SO
4 2-, then white precipitate is scattered in water, adds the aqueous hydrogen peroxide solution (Ti of 30%
4+and H
2o
2mol ratio be 1:4) stir and make precipitation peptization, form yellow pertitanic acid (PTA) colloidal sol after stablizing 15 hours, PTA colloidal sol is dried at 75 DEG C and is formed xerogel, within 1 hour, obtains nano titanium dioxide powder after grinding with 500 DEG C of calcinings;
(3) liquid microelement preparation: by ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2o is soluble in water obtains liquid microelement, wherein ZnSO
47H
2o, FeSO
47H
2o, CuSO
45H
2o and MnSO
4h
2content 0.35g/L, 0.3g/L, 0.24g/L and the 0.18g/L respectively of O;
(4) lipopeptid-nano titanium oxide compound formulation obtains: in liquid microelement, add lipopeptid and nano titanium oxide successively, wherein, the content of lipopeptid is 0.1%, the content of nano titanium oxide is 0.25%, after stirring shakes up on shaking table with 30 DEG C, 170rpm vibrates 24h, namely obtains lipopeptid-nano titanium oxide compound formulation.
Embodiment 4:
Ultra-pure water, titanium sulfate (chemical pure), ammoniacal liquor (analyzing pure), hydrogen peroxide (analyzing pure) and sulphate (analyzing pure) is adopted to obtain lipopeptid, nano titanium oxide, liquid microelement and lipopeptid-nano titanium oxide compound formulation according to the preparation method of embodiment 3.
The preparation of 1, nano titanium oxide-micro-complex liquid
In liquid microelement, add nano titanium oxide, to stir after shaking up with 30 DEG C on shaking table, 170rpm vibrates 24h, namely obtains nano titanium oxide-micro-complex liquid.In this complex liquid, the mass percent of nano titanium oxide is 0.25%.
2, structural characterization
2.1X-x ray diffraction (XRD) is analyzed:
Japanese Rigaku company D/MAX-2500/PCX-x ray diffractometer x is used to analyze nano titanium dioxide powder.As shown in Figure 1, consult PDF card JCPDS21-1272, the characteristic peak at 25.3 ° that XRD collection of illustrative plates marks, 37.8 °, 48.0 °, 53.8 ° and 62.8 ° (2 θ) places distinguishes the crystal face of corresponding anatase (101), (004), (200), (105) and (204), simultaneously locate not existing characteristics peak and show that brockite and rutile are not present in sample for 27.4 ° and 30.8 °, result shows that the nano titanium dioxide powder prepared is Detitanium-ore-type.
2.2 transmission electron microscopes (TEM) and SEM (SEM) are analyzed:
Japanese JEOL company JEM-2100F200kV transmission electron microscope and HIT S-4800 field emission scanning electron microscope is used to carry out observation and analysis to the appearance and size of nano titanium dioxide powder.As shown in Figure 2 and Figure 3, nano titanium dioxide powder is formed by the nano particle gathering of many 10 ~ 40nm.
2.3 infrared spectrums (FT-IR) are analyzed:
After nano titanium oxide-micro-complex liquid and lipopeptid-nano titanium oxide compound formulation carry out centrifugation respectively, with milli-Q water precipitation several, then dry at 60 DEG C, by nano titanium oxide-micro-complexing agent, lipopeptid-nano titanium oxide compound formulation powder and nano titanium dioxide powder KBr compressing tablet after grinding, ThermoFisherIS10 infrared spectrometer is adopted to analyze the change of main functional group, wave-number range 500 ~ 4000cm
-1.
As shown in Figure 4, curve (a) is more known with (b), and the powder that nano titanium oxide-micro-complex liquid obtains after process is at 3083cm
-1and 3730cm
-1having there is new peak in place, is the characteristic absorption of O-H group, but 3083cm
-1the peak at place is the characteristic absorption of the O-H group that hydrogen bond connects, simultaneously 476cm
-1and 1135cm
-1the characteristic absorption peak of Ti-O-Ti and Ti-O at place has been displaced to 482cm respectively
-1and 1125cm
-1place, shows that nano titanium oxide-micro-complex liquid medium trace element ion and nano titanium oxide combine.
Curve (a) is more known with (c), and the powder that lipopeptid-nano titanium oxide compound formulation obtains after process is at 1457cm
-1, 1542cm
-1, 1649cm
-1, 1717cm
-1, 2851cm
-1, 2921cm
-1, 3188cm
-1, 3621cm
-1, 3735cm
-1having there is new peak in place, is respectively C=O, CH2 in CH3, acid amides bands of a spectrum II (N-H or C-N), acid amides bands of a spectrum I (C=O), ketone, the O-H of hydrogen bond connection, the characteristic absorption of other O-H, simultaneously 476cm
-1and 1135cm
-1the characteristic absorption peak of Ti-O-Ti and Ti-O at place has been displaced to 468cm respectively
-1and 1122cm
-1place, shows that in lipopeptid-nano titanium oxide compound formulation, lipopeptid, trace element ion and nano titanium oxide combine.
Embodiment 5: lipopeptid and lipopeptid-nano titanium oxide compound formulation detect the inhibit activities of bacterial wilt of tomato pathogen.
1, bacterial wilt of tomato pathogen bacterium solution preparation.
Picking bacterial wilt of tomato pathogen on TTC flat board, in access liquid NA medium, 30 DEG C, 170rpm shaking table is cultured to logarithmic phase, with sterile water, bacteria suspension is diluted to 10
6~ 10
7cFU/mL, pathogen bacteria suspension is used in obtained test.
2, inhibit activities detects.By nano titanium dioxide powder 121 DEG C, 20min sterilizing, it is degerming that liquid microelement crosses 0.22 μm of filter membrane after adding lipopeptid, aseptically prepares lipopeptid-nano titanium oxide compound formulation.Pathogen bacteria suspension 200 μ L is spread evenly across on NA culture medium flat plate, then 100 μ L variable concentrations lipopeptide solution (200mg/L are drawn, 500mg/L, 750mg/L, 1000mg/L, 1500mg/L, 2000mg/L) inject dull and stereotyped upper Oxford cup, cultivate 24h, observe antibacterial situation and measure inhibition zone size, repeating 3 times for 30 DEG C.As shown in table 1 and Fig. 5, lipopeptid has stronger inhibition to bacterial wilt of tomato pathogen, and inhibition zone is obvious.
Aseptically preparation is 10 containing bacteria suspension concentration
6powder (0 after the pathogen of CFU/mL and the lipopeptid of variable concentrations-nano titanium oxide compound formulation centrifugal drying, 0.15mg/mL, 0.3mg/mL, 0.6mg/mL, 1.2mg/mL, 2.4mg/mL, NA liquid nutrient medium test tube 4.8mg/mL), under dark or illumination condition 30 DEG C, 170rpm shaking table cultivates 24 hours, measures the minimal inhibitory concentration (MIC) of powder.Result shows, under dark or illumination condition, the MIC of the powder after lipopeptid-nano titanium oxide compound formulation centrifugal drying is 1.2mg/mL.
Table 1 variable concentrations lipopeptide solution is to the bacteriostasis of bacterial wilt of tomato pathogen
Embodiment 6:
Adopt ultra-pure water, titanium sulfate (chemical pure), ammoniacal liquor (analyzing pure), hydrogen peroxide (analyzing pure) and sulphate (analyzing pure) to obtain lipopeptid, nano titanium oxide, liquid microelement and lipopeptid-nano titanium oxide compound formulation according to the preparation method of embodiment 3, obtain nano titanium oxide-micro-complex liquid according to the preparation method of embodiment 4.
The preparation of 1, lipopeptid-micro-complex liquid
In liquid microelement, add lipopeptid, to stir after shaking up with 30 DEG C on shaking table, 170rpm vibrates 24h, namely obtains lipopeptid-micro-complex liquid.In this complex liquid, the mass percent of lipopeptid is 0.1%.
2, lipopeptid-nano titanium oxide compound formulation is to the greenhouse controlling experiment of bacterial wilt of tomato
Controlling experiment carries out in Jiangsu Province's solid organic castoff resource Hi-Tech Key Laboratory green house, 5 process are established in experiment, scheme is as follows: (1) Control, gets 10ml ultra-pure water and evenly sprays blade face in every strain tomato seedling and basal part of stem, in contrast; (2) MS, gets 10ml liquid microelement and evenly sprays blade face in every strain tomato seedling and basal part of stem; (3) LFA, gets 10ml lipopeptid-micro-complex liquid and evenly sprays blade face in every strain tomato seedling and basal part of stem; (4) LFB, gets 10ml nano titanium oxide-micro-complex liquid and evenly sprays blade face in every strain tomato seedling and basal part of stem; (5) LFC, gets 10ml lipopeptid-nano titanium oxide compound formulation and evenly sprays blade face in every strain tomato seedling and basal part of stem.Each process 30 basin, every basin dress 2kg soil, applies equivalent nitrogen, phosphorus, potassium fertilizer (comprising: urea, superphosphate, potassium chloride), every pot transplanting 1 strain tomato seedling.Transplant and in soil, pour into bacterial wilt of tomato pathogen bacteria suspension after 7 days, make pathogen concentration reach 10
6more than CFU/g soil, fill with bacterium, after 24 hours, process is carried out 1 time by above-mentioned 5 processing schemes to tomato seedling.
Within 4th, 8,12,16,20,24,28 day, press severity Scaling standard after treatment, record incidence, adds up each process disease index (DI) and control efficiency, and statistical disposition respectively processes plant above ground portion, underground part dry weight in 28 days afterwards.
Severity Scaling standard and disease index computational methods as follows:
0 grade: plant is normal; 1 grade: the blade of plant less than 25% shows as wilting symptom;
2 grades: the blade of plant 26% ~ 50% shows as wilting symptom;
3 grades: the blade of plant 51% ~ 75% shows as wilting symptom;
4 grades: plant more than 76% blade shows as wilting symptom or plant is dead.
Disease index (DI)=[∑ (each sick level plant number × at different levels represent numerical value)]/(investigating total strain number × highest level numerical value) × 100%
Control efficiency=(contrast disease index-process disease index)/contrast disease index × 100%
3, lipopeptid-nano titanium oxide compound formulation is to the greenhouse growth-promoting test of bacterial wilt of tomato
Growth-promoting test is carried out in Jiangsu Province's solid organic castoff resource Hi-Tech Key Laboratory green house, 5 process are established in experiment, scheme is as follows: (1) Control, gets 10ml ultra-pure water and evenly sprays blade face in every strain tomato seedling and basal part of stem, in contrast; (2) MS, gets 10ml liquid microelement and evenly sprays blade face in every strain tomato seedling and basal part of stem; (3) LFA, gets 10ml lipopeptid-micro-complex liquid and evenly sprays blade face in every strain tomato seedling and basal part of stem; (4) LFB, gets 10ml nano titanium oxide-micro-complex liquid and evenly sprays blade face in every strain tomato seedling and basal part of stem; (5) LFC, gets 10ml lipopeptid-nano titanium oxide compound formulation and evenly sprays blade face in every strain tomato seedling and basal part of stem.Each process 15 basin, every basin dress 2kg soil, applies equivalent nitrogen, phosphorus, potassium fertilizer (comprising: urea, superphosphate, potassium chloride), every pot transplanting 1 strain tomato seedling.Transplant after 7 days and process is carried out 1 time by above-mentioned 5 processing schemes to tomato seedling.Process after 30 days, add up that each process plant plant height, stem are thick, SPAD value and overground part underground part dry weight and fresh weight of plant seedlings.
4, results and analysis:
As shown in table 2 and Fig. 6, along with the growth of tomato plant, compared with other process, take the disease index spraying lipopeptid-nano titanium oxide compound formulation very low, after process, when the 28th day, relative control efficiency is 67.47%, control efficiency compared to other process has significant difference, illustrate that the combination of nano titanium oxide and lipopeptid in lipopeptid-nano titanium oxide compound formulation can improve the stability of lipopeptid better, extend action time of lipopeptid, this lipopeptid-nano titanium oxide compound formulation can control of plant bacterial wilt effectively.
As shown in Figure 7 and Figure 8, after processing 28 days in greenhouse controlling experiment, plant above ground portion and the underground part dry weight of taking to spray lipopeptid-nano titanium oxide compound formulation are all high than other process, and compared with the control there is significant difference, illustrate that this lipopeptid-nano titanium oxide compound formulation can promote tomato plant strain growth while control of plant bacterial wilt.
As shown in table 3 and Fig. 9, in greenhouse growth-promoting test, process is after 30 days, take to spray that the plant plant height of lipopeptid-nano titanium oxide compound formulation, stem are thick, SPAD value and the fresh and dried weight average of overground part underground part higher than other process, and except SPAD value, other values all have significant difference compared with Control, illustrate that this lipopeptid-nano titanium oxide compound formulation can promote tomato plant strain growth, compared with other processing modes, taking to spray lipopeptid-nano titanium oxide compound formulation can promote tomato plant strain growth better.
Table 2 greenhouse controlling experiment different disposal is to the control efficiency of bacterial wilt of tomato
Note: different lowercase represents significant difference (p<0.05)
Thick and the SPAD value of the plant height of each process plant in the test of table 3 greenhouse growth-promoting, stem
Note: different lowercase represents significant difference (p<0.05).
Claims (10)
1. lipopeptid-nano titanium oxide compound formulation, is characterized in that, said preparation comprises the component of following mass percent:
Lipopeptid 0.075% ~ 0.15%;
Nano titanium oxide 0.2% ~ 0.3%;
Trace elements zn
2+0.0069% ~ 0.0092%, Fe
2+0.005% ~ 0.007%, Cu
2+0.0052% ~ 0.0091% and Mn
2+0.005% ~ 0.0083%;
All the other are water.
2. lipopeptid according to claim 1-nano titanium oxide compound formulation, is characterized in that, described lipopeptid, is prepared as follows and obtains:
(1a) activate bacillus amyloliquefaciens, obtain seed liquor;
(1b) be transferred in fermentation medium by seed liquor, regulate relative moisture in fermentation tank to be greater than 80%, lipopeptid is produced in fermentation;
(1c), after fermentation ends, the mixture obtained fermenting and water mix according to 1:10 (w/v, g/mL) ratio, centrifugal, collect supernatant, regulate supernatant pH to be 1 ~ 3 with hydrochloric acid, leave standstill, centrifugal again, collecting precipitation, precipitation uses carrene extracting, then revolves and boil off except carrene, residue is by water-soluble solution and adjust pH to be 7.0, and last freeze drying obtains lipopeptid powder.
3. lipopeptid according to claim 1-nano titanium oxide compound formulation, is characterized in that, described nano titanium oxide is prepared as follows and obtains:
(2a) to press the concentration of 0.1mol/L soluble in water for titanium sulfate, and add the ammonia spirit of 2 ~ 3mol/L, the pH making solution is 6 ~ 8, obtains mixture;
(2b) mixture is centrifugal, collecting precipitation, washes precipitation with water;
(2c) precipitation that step (2b) obtains is scattered in water, adds the aqueous hydrogen peroxide solution that volume ratio is 30%, make Ti in solution
4+and H
2o
2mol ratio be 1:3 ~ 5, stir, leave standstill 15 ~ 20 hours, obtain peroxo titanic acid sol;
(2d) peroxo titanic acid sol is dried formation xerogel at 60 ~ 75 DEG C, by xerogel grind into powder, calcine 1 ~ 2 hour under 400 ~ 600 DEG C of conditions, obtain nano titanium dioxide powder.
4. lipopeptid according to claim 1-nano titanium oxide compound formulation, is characterized in that, described trace element is Zn
2+salt, Fe
2+salt, Cu
2+salt and Mn
2+any one or a few mixture in salt.
5. lipopeptid according to claim 4-nano titanium oxide compound formulation, is characterized in that, described trace element is Zn
2+salt, Fe
2+salt, Cu
2+salt and Mn
2+the mixture of salt.
6. the preparation method of lipopeptid according to claim 1-nano titanium oxide compound formulation, it is characterized in that, the method comprises the steps:
(1) preparation of lipopeptid:
(1a) activate bacillus amyloliquefaciens, obtain seed liquor;
(1b) be transferred in fermentation medium by seed liquor, regulate relative moisture in fermentation tank to be greater than 80%, lipopeptid is produced in fermentation;
(1c) after fermentation ends, by fermenting, the mixture obtained mixes according to 1g:10mL ratio with water, centrifugal, collects supernatant, supernatant pH is regulated to be 1 ~ 3 with hydrochloric acid, leave standstill, more centrifugal, collecting precipitation, precipitation uses carrene extracting, revolve and boil off except carrene, residue is by water-soluble solution and adjust pH to be 7.0, and last freeze drying obtains lipopeptid powder;
(2) preparation of nano titanium oxide:
(2a) to press the concentration of 0.1mol/L soluble in water for titanium sulfate, and add the ammonia spirit of 2 ~ 3mol/L, the pH making solution is 6 ~ 8, obtains mixture;
(2b) mixture is centrifugal, collecting precipitation, washes precipitation with water;
(2c) precipitation that step (2b) obtains is scattered in water, adds the aqueous hydrogen peroxide solution that volume ratio is 30%, make Ti in solution
4+and H
2o
2mol ratio be 1:3 ~ 5, stir, leave standstill 15 ~ 20 hours, obtain peroxo titanic acid sol;
(2d) peroxo titanic acid sol is dried formation xerogel at 60 ~ 75 DEG C, by xerogel grind into powder, calcine 1 ~ 2 hour under 400 ~ 600 DEG C of conditions, obtain nano titanium dioxide powder;
(3) preparation of liquid microelement: be dissolved in the water by trace element, obtains trace element water-soluble liquid;
(4) lipopeptid-nano titanium oxide compound formulation obtains: in the trace element water-soluble liquid that step (3) obtains, add lipopeptid that step (1) obtains and the nano titanium oxide that step (2) obtains successively, stirring shakes up, 20 ~ 40 DEG C, under 150 ~ 250rpm condition, vibration 10 ~ 30h, namely obtains lipopeptid-nano titanium oxide compound formulation.
7. preparation method according to claim 6, it is characterized in that, in step (1b), described fermentation medium as follows equal proportion prepares: take 4.5 ~ 6kg analysis for soybean powder, 3 ~ 4.5kg stalk, 0.15 ~ 0.2kg starch and 0.15 ~ 0.2kg yeast extract, put into fermentation tank, add 1 ~ 2L inorganic salt solution, pH is regulated to be 7 ~ 8, the moisture content regulating medium is 40% ~ 60%, and described inorganic salt solution, its formula is: 1.0g/LKH
2pO
4, 0.5g/LMgSO
47H
2o, 0.5g/LKCl, 2 × 10
-3g/LL-phenyl alanine, 5 × 10
-3g/LMnSO
4, 0.16 × 10
-3g/LCuSO
4, 0.15 × 10
- 3g/LFeSO
4.
8. preparation method according to claim 6, it is characterized in that, in step (1b), fermentation process adopts temperature-variable fermentation, keeps that throughput is 0.1 ~ 0.5vvm, tank pressure is 0.01 ~ 0.05Mpa during the fermentation, and first stage fermentation temperature is 25 ~ 40 DEG C, fermentation 20 ~ 24h, the fermentation temperature of second stage is 28 ~ 40 DEG C, fermentation 16 ~ 24h, and ensures that the temperature of second stage fermentation is higher than first stage temperature 0 ~ 7 DEG C.
9. the lipopeptid according to claim 1-application of nano titanium oxide compound formulation in control of plant bacterial wilt.
10. lipopeptid according to claim 1-nano titanium oxide compound formulation is promoting the application on tomato plant strain growth.
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