CN105969828A - Preparation method of trichoderma harzianum strain antimicrobial peptide and application to China rose black spot prevention - Google Patents
Preparation method of trichoderma harzianum strain antimicrobial peptide and application to China rose black spot prevention Download PDFInfo
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- CN105969828A CN105969828A CN201610363304.3A CN201610363304A CN105969828A CN 105969828 A CN105969828 A CN 105969828A CN 201610363304 A CN201610363304 A CN 201610363304A CN 105969828 A CN105969828 A CN 105969828A
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- 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K1/16—Extraction; Separation; Purification by chromatography
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Abstract
The invention provides a preparation method of a trichoderma harzianum strain antimicrobial peptide. A 1*10<6> CFU/mL trichoderma harzianum spore suspension is inoculated in a liquid culture medium, wherein a trichoderma harzianum strain is selected from one of strains with the numbers of CGMCC 3.5488, CGMCC 3.6635 and CGMCC 3.5492; the mixture is placed in a shaker to be cultured for 48-96 h; a bacterial solution is transferred into a fermentation tank to be fermented and cultured for 6-8 days; after fermented liquor is filtered, methyl alcohol and water serve as a mobile phase, crude separation is conducted by means of a macroporous resin column, then a crude separation product is purified with methyl alcohol as a mobile phase by means of a sephadex column, the purified product is frozen and dried, and the trichoderma harzianum strain antimicrobial peptide is obtained. The invention further provides application of trichoderma harzianum strain antimicrobial peptide powder prepared through the method to China rose black spot prevention. The prepared trichoderma harzianum strain antimicrobial peptide is high in purity, and has a high prevention effect on China rose black spot.
Description
Technical field
The invention belongs to field of biology, relate to a kind of Chinese rose, specifically a kind of T. harzianum strains antibacterial peptide
Preparation method and preventing and treating Chinese Rose in application.
Background technology
Chinese rose (Rosa chinensisJacp.), also known as Fols Rosae, Herba Polygoni Capitati etc., pattern is beautiful, and the four seasons are normally opened, have
The good reputation " spending middle queen ", is the most important ornamental flower.The whole nation has 38 cities using Chinese rose as city flower at present.Month
Season and companion flowers Flos Rosae Rugosae thereof all can extract quintessence oil, are widely used in cosmetics and food service industry, are worth higher than gold.But Chinese rose
Often occurring some diseases to have influence on its ornamental value and medical value in planting process, Chinese Rose is exactly the most normal
See that there is most destructive a kind of disease.Chinese Rose infectiousness is strong and preventing and treating is difficult.The pathogen of Chinese Rose is Flos Rosae Multiflorae
Marssonina [Marssonina rosae(Lib.) Died.], site of pathological change is mainly at blade, and scab is circular, and edge radiates
Shape, around scab, mesophyll turns yellow, and sick leaf is easy to fall off.Time serious, the most several scabs are connected, around large area jaundice, even plant
Middle and lower part blade is the most withered and the most yellow to come off, and only leaves several, top young leaves, shaft-like in light.Melasma makes plant strain growth gesture weaken, weight
Person causes death, and growth and outward appearance to Chinese rose cause and have a strong impact on, and therefore reduce Chinese rose ornamental values and the economic values.
Up to now, China's most Chinese rose growing area all can have the generation of this disease, destructive extremely strong.
Chemical bactericide has considerable influence, and the Drug resistance day of phytopathogen to ecological environment, biology and human body
Benefit increases, and biological control agents therefore can be utilized to prevent and treat floral crops diseases as biofungicide, to alleviate chemical pesticide to ring
Environment pollution.Trichoderma (TrichodermaSpp) since being found phytopathogen is had inhibitory action, always as important
The biocontrol of plant disease factor and receive much attention.Trichoderma spp. is a kind of Hyperparasite, its mycelia often through with host mycelia
Parallel Growth, the mode parasitism such as tend to grow, identify, contact, be wound around, adhere to, penetrate and stretch in host mycelia, directly from posting
Draw nutrition in main mycelia, thus destroy the growth promoter of host, make host mycelia lose the globality of its protoplast and break
Split or be only left ghost, until dead.Utilize trichoderma to carry out Biological control and early have application, but be all directly to use sending out of trichoderma
The mode that ferment product directly sprays carrys out controlling plant diseases.Due to the tunning complicated component of trichoderma, it is prevented and treated plant
The mechanism of disease is the most unclear.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of trichoderma harzianum antibacterial peptide and at preventing and treating Chinese Rose
In application, the preparation method of described this trichoderma harzianum antibacterial peptide and the application in preventing and treating Chinese Rose to solve
By the technical problem of the poor effect of the tunning preventing and treating Chinese Rose of trichoderma in prior art.
The invention provides the preparation method of a kind of trichoderma harzianum antibacterial peptide, comprise the steps:
1) by 1 × 106CFU/mL trichoderma harzianum spore suspension is inoculated in fluid medium, described T. harzianum strains
Derive from China General Microbiological culture presevation administrative center, selected from strain number be CGMCC 3.5488, CGMCC3.6635,
Or any one in CGMCC 3.5492, described trichoderma harzianum spore suspension and the volume ratio of fluid medium are
1ml: 80~120ml；Described fluid medium is made up of water, Rhizoma Solani tuber osi and glucose, in every liter of fluid medium, and described soil
Bean is 180 ~ 220 g, and described glucose is 18 ~ 22g, and surplus is water, is placed in 25 ~ 30 DEG C, in the shaking table of 150 ~ 200r/min
Cultivate 48 ~ 96h；
2) bacterium solution is transferred in fermentation tank, in 25 ~ 30 DEG C, fermentation culture 6 under conditions of stir speed (S.S.) 150 ~ 200r/min ~
8 days, described fermentation medium by Rhizoma Solani tuber osi, sucrose, glucose, anhydrous sodium acetate, Magnesium sulfate heptahydrate, peptone, manganese chloride,
Defoamer and water composition, the concentration of above-mentioned substance is: Rhizoma Solani tuber osi 200 g/L, sucrose 10 g/L, glucose 10 g/L, anhydrous acetic acid
Sodium 1.66 g/L, Magnesium sulfate heptahydrate 5. 29 g/L, peptone 0. 5 g/L, manganese chloride 0. 15 g/L, defoamer 0.05
G/L, surplus is water；
3) by step 2) filtering fermentation liquor after, with methanol-water make flowing phase, use macroporous adsorptive resins carry out crude separation,
Then being done with methanol by crude separation product and flow through polydextran gel column purification, purified product carries out lyophilization and obtains Ha Ci
Trichoderma antibacterial peptide.
Further, described defoamer is polypropylene glycerol aether or polyoxyethylene polyoxypropylene glycerin ether.
Present invention also offers trichoderma harzianum antibacterial peptide answering in preventing and treating Chinese Rose prepared by above-mentioned method
With.
Present invention also offers a kind of method preventing and treating Chinese Rose, comprise the steps:
1) step processing soil；By the trichoderma harzianum antibacterial peptide prepared by above-mentioned steps and seedling medium with 1:
The ratio of 500 is sufficiently mixed；
2) step that a root processes, before kind of transplantation of seedlings, by dilute for the trichoderma harzianum antibacterial peptide prepared by above-mentioned steps
Root is soaked 20 ~ 40 minutes, then field planting after releasing 400 ~ 600 times；After transplanting, by antibacterial for the trichoderma harzianum prepared by above-mentioned steps
Pouring root after peptide dilution 400 ~ 600 times；
3) step of a spraying；Carry out after the trichoderma harzianum antibacterial peptide prepared by above-mentioned steps is diluted 600～800 times
Spraying.
Further, in step 1), by by the trichoderma harzianum antibacterial peptide of above-mentioned preparation and seedling medium with 1:10
Ratio be sufficiently mixed after, be sprinkled into seedbed, per kilogram processes 10 15 square metres of seedbed.
Trichoderma harzianum (Trichoderma harzianum) is a kind of in trichoderma important to prevent and treat phytopathy
The fungus of former bacterium.Trichoderma harzianum fermented liquid can be used for the preventing and treating of various plants fungal disease.Wherein rise main work for Trichoderma spp.
Bacterium antibacterial peptide.The trichoderma harzianum that the present invention selects is from China General Microbiological culture presevation administrative center
(www.cgmcc.net), strain number is CGMCC 3.5488, CGMCC3.6635, CGMCC 3.5492, according to this website and
The bulletin of administrative center, any can buy above-mentioned three kind bacterial strain per capita.
Trichoderma harzianum antibacterial peptide has antifungal, the biological activity of anti-gram-positive bacteria widely, and it is thin that it has
After birth rhetorical function, forms voltage-dependent or independent ion channel or hole, increase pair in nature and artificial bilayer membrane
The permeability etc. of molecular layer lipid film, to kill phytopathogen.The cell wall that trichoderma antibacterial peptide produces with fungus Trichoderma
Digestive enzyme synergy has coordinate repression effect to plant pathogenic fungi.
The present invention compares with prior art, and it has the technical effect that positive and obvious.Trichoderma harzianum prepared by the present invention
Antibacterial peptide purity is high, has Chinese Rose the highest to prevention effect.Trichoderma harzianum antibacterial peptide prepared by the present invention is micro-life
Thing preparation, environmentally friendly, harmless, and also easy to use, the morbidity of Chinese Rose can be alleviated, reduce the state of an illness and refer to
Number, protection effect, more than 90%, significantly inhibits effect to Chinese Rose.
Accompanying drawing explanation
Fig. 1 is the photo before ' Meath, pula ' modern rose cultivars uses.
Fig. 2 sprays rear photo for ' Meath, pula ' modern rose cultivars.
Fig. 3 is the photo before ' Du Kesa ' modern rose cultivars uses.
Fig. 4 is the photo after ' Du Kesa ' modern rose cultivars sprays.
Detailed description of the invention
Below in conjunction with preferred embodiment, technical scheme is set forth in.Following example are used only for
The description and interpretation present invention, and do not constitute the restriction to technical solution of the present invention.
Prepared by embodiment 1 trichoderma harzianum antibacterial peptide sample
1. by 1mL 1 × 106CFU/mL trichoderma harzianum CGMCC 3.5488 or CGMCC 3.6635 or CGMCC 3.5492 spore
Fullness over the chest during pregnancy liquid is inoculated in 100 mL fluid mediums (Rhizoma Solani tuber osi 200 g/L, glucose 20 g/L), is placed in 28 DEG C, 180 r/
72 h cultivated by min shaking table；
The most then bacterium solution is transferred in fermentation tank (7 L), in 28 DEG C, stir speed (S.S.) 180 r/min, fermentation culture 7
d.Fermentation medium is Rhizoma Solani tuber osi 200 g/L, sucrose 10 g/L, glucose 10 g/L, anhydrous sodium acetate 1.66 g/L, seven water sulfur
Acid magnesium 5.29 g/L, peptone 0.5 g/L, manganese chloride 0.15 g/L, defoamer 0.05 g/L；
After 3.1 L fermentation liquor filtered through gauze, weighing for dry, 4 L fermentation liquids are used as to flow mutually with methanol-water
XAD-2 macroporous adsorptive resins carries out crude separation, then with methanol, target product is done flowing and coagulates through LH-20 glucosan
Glue column purification, purified product carries out lyophilization and obtains trichoderma harzianum antibacterial peptide powder goods.
The embodiment 2 trichoderma harzianum antimicrobial peptide preparation prevention and controls to Chinese Rose
1) soil is processed
Sowing or cuttage substrate can be by the trichoderma harzianum antibacterial peptide powder of preparation and seedling medium with the ratios of mass ratio 1:500
Example directly uses after being sufficiently mixed；If directly processing seedbed, can be sprinkled into after ratio and the seedling medium of 1:10 in mass ratio mix
Seedbed, per kilogram processes 10 15 squares of seedbed；
2) root processes
Before planting transplantation of seedlings, with 500 times of liquid (mass percent concentrations of trichoderma harzianum antibacterial peptide of trichoderma harzianum antibacterial peptide
It is 0.2%) soak root 30 minutes, then field planting；After transplanting, (mass percent of trichoderma harzianum antibacterial peptide is dense for available 500 times of liquid
Degree is 0.2%) pouring root.
3) spraying
Spray with 800 times of liquid (trichoderma harzianum antibacterial peptide is diluted 800 times) of trichoderma harzianum antibacterial peptide, annual
The 3-4 month, each week sprays 1 time, and premorbid or initial stage using effect are more preferably.
Preliminary test using effect contrast in Expo shop Chinese Rose prevents, can by Fig. 1, Fig. 2, Fig. 3 and Fig. 4
Knowing, through the prevention of trichoderma harzianum antibacterial peptide, statistics understands, and the blade ratio infecting melasma declines 80%.
Claims (5)
1. the preparation method of a trichoderma harzianum antibacterial peptide, it is characterised in that comprise the steps:
1) by 1 × 106CFU/mL trichoderma harzianum spore suspension is inoculated in fluid medium, described T. harzianum strains
Derive from China General Microbiological culture presevation administrative center, selected from strain number be CGMCC 3.5488, CGMCC3.6635,
Or any one in CGMCC 3.5492, described trichoderma harzianum spore suspension and the volume ratio of fluid medium are
1ml: 80~120ml；Described fluid medium is made up of water, Rhizoma Solani tuber osi and glucose, in every liter of fluid medium, and described soil
Bean is 180 ~ 220 g, and described glucose is 18 ~ 22 g, and surplus is water, is placed in 25 ~ 30 DEG C, the shaking table of 150 ~ 200r/min
Middle cultivation 48 ~ 96h；
2) bacterium solution is transferred in fermentation tank, in 25 ~ 30 DEG C, fermentation culture 6 under conditions of stir speed (S.S.) 150 ~ 200r/min ~
8 days, described fermentation medium by Rhizoma Solani tuber osi, sucrose, glucose, anhydrous sodium acetate, Magnesium sulfate heptahydrate, peptone, manganese chloride,
Defoamer and water composition, the concentration of above-mentioned substance is: Rhizoma Solani tuber osi 200 g/L, sucrose 10 g/L, glucose 10 g/L, anhydrous acetic acid
Sodium 1.66 g/L, Magnesium sulfate heptahydrate 5. 29 g/L, peptone 0. 5 g/L, manganese chloride 0. 15 g/L, defoamer 0.05
G/L, surplus is water；
3) by step 2) filtering fermentation liquor after, with methanol-water make flowing phase, use macroporous adsorptive resins carry out crude separation,
Then being done with methanol by crude separation product and flow through polydextran gel column purification, purified product carries out lyophilization and obtains Ha Ci
Trichoderma antibacterial peptide.
The preparation method of a kind of trichoderma harzianum antibacterial peptide the most according to claim 1, it is characterised in that: described froth breaking
Agent is polypropylene glycerol aether or polyoxyethylene polyoxypropylene glycerin ether.
3. the trichoderma harzianum antibacterial peptide prepared by the method described in claim 1 application in preventing and treating Chinese Rose.
4. the method preventing and treating Chinese Rose, it is characterised in that comprise the steps:
1) step processing soil；Trichoderma harzianum antibacterial peptide claim 1 prepared and seedling medium are with mass ratio
Ratio for 1:500 is sufficiently mixed；
2) step that a root processes, before kind of transplantation of seedlings, trichoderma harzianum antibacterial peptide dilution prepared by claim 1
Root is soaked 20 ~ 40 minutes, then field planting after 400 ~ 600 times；After transplanting, trichoderma harzianum antibacterial peptide claim 1 prepared is dilute
Pouring root after releasing 400 ~ 600 times；
3) step of a spraying；Trichoderma harzianum antibacterial peptide claim 1 prepared sprays after diluting 600～800 times
Mist.
A kind of method preventing and treating Chinese Rose the most according to claim 4, it is characterised in that: in step 1), will power
After profit requires that the trichoderma harzianum antibacterial peptide of 1 preparation and seedling medium are sufficiently mixed with the ratio of 1:10, it is sprinkled into seedbed, per kilogram
Process 10 15 square metres of seedbed.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610363304.3A CN105969828A (en) | 2016-05-27 | 2016-05-27 | Preparation method of trichoderma harzianum strain antimicrobial peptide and application to China rose black spot prevention |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107242123A (en) * | 2017-06-21 | 2017-10-13 | 磐安县派普特生物科技有限公司 | A kind of Rapid Rooting method of Chinese rose cuttage |
| CN109401985A (en) * | 2018-10-08 | 2019-03-01 | 山东大学 | The Δ Tlrgt2 trichoderma engineering bacteria and its construction method of one plant height production peptaibols antibacterial peptide and application |
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| CN101057593A (en) * | 2007-01-26 | 2007-10-24 | 中国计量学院 | Preparation method for preventing and treating vegetable fungicide microbial pesticides |
| CN103045482A (en) * | 2011-10-11 | 2013-04-17 | 上海万力华生物科技有限公司 | Trichoderma harzianum strain |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101057593A (en) * | 2007-01-26 | 2007-10-24 | 中国计量学院 | Preparation method for preventing and treating vegetable fungicide microbial pesticides |
| CN103045482A (en) * | 2011-10-11 | 2013-04-17 | 上海万力华生物科技有限公司 | Trichoderma harzianum strain |
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| 张华江: "《食品添加剂原理与应用》", 31 August 2014, 中国农业出版社 * |
| 张宝俊: "《抗病微生物的研发及应用》", 31 July 2013 * |
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| 潘顺: "哈茨木霉发酵液中peptaibols抗茵肽的鉴定及活性研究", 《中国生物防治学报》 * |
| 潘顺: "高效液相色谱-质谱联用法高通量筛选及检测哈茨木霉菌发酵液中peptaibols类抗菌肽", 《中国药学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107242123A (en) * | 2017-06-21 | 2017-10-13 | 磐安县派普特生物科技有限公司 | A kind of Rapid Rooting method of Chinese rose cuttage |
| CN109401985A (en) * | 2018-10-08 | 2019-03-01 | 山东大学 | The Δ Tlrgt2 trichoderma engineering bacteria and its construction method of one plant height production peptaibols antibacterial peptide and application |
| CN109401985B (en) * | 2018-10-08 | 2021-10-26 | 山东大学 | Delta Tlrgt2 trichoderma engineering bacterium for high yield of peptaibols antibacterial peptide and construction method and application thereof |
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Application publication date: 20160928 |