CN108739864A - Plant endogenesis bacteria pesticide and preparation method thereof - Google Patents
Plant endogenesis bacteria pesticide and preparation method thereof Download PDFInfo
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- CN108739864A CN108739864A CN201810620553.5A CN201810620553A CN108739864A CN 108739864 A CN108739864 A CN 108739864A CN 201810620553 A CN201810620553 A CN 201810620553A CN 108739864 A CN108739864 A CN 108739864A
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- plant
- endophyte
- endogenesis
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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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- 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
Abstract
Plant endogenesis bacteria pesticide preparation method, including step：Endophyte of plant is extracted from Celastraceae plant；The endophyte of plant of purifying culture extraction is in case test；It is sprayed on drosophila and mosquito respectively after the metabolite of the endophyte of plant of culture is diluted, selects high lethality plant endogenesis bacteria strain；The bacterial strain selected is subjected to fermented and cultured；It filters and the zymotic fluid for concentrating fermented and cultured obtains plant endogenesis bacteria pesticide.Insecticide prepared by the plant endogenesis bacteria pesticide preparation method of the present invention, endophyte of plant screened itself is extracted from the plant with anthelminthic effect, good to pest controling effect, safe and non-toxic to people, animal, plant, free from environmental pollution, noresidue；Endophyte of plant kills high specificity to disease pest, while not injuring natural enemy and beneficial organism, ecological natural can be kept to balance；Raw materials for production and active ingredient belong to natural products, can back to nature and reduce for naturally destroying, ensure sustainable development.
Description
Technical field
The present invention relates to the preparations of microbial pesticide more particularly to plant endogenesis bacteria pesticide and preparation method thereof.
Background technology
China's pesticide product unreasonable structure at present, organophosphorus insecticide accounts for 70% in insecticidal total yield, and high
Drugs kind accounts for organic phosphorous insecticide 70%.High poison is wide in variety in China's insecticide, yield is big, it has also become pesticide producing, use
Conspicuous contradiction.Chemical pesticide remains in water, soil, animal and the body of the mankind, strangles the vigor of nature, destroys ecology
Environment, with increasing for chemicals, the harm aggravation to the mankind.Organism pesticide refers to be had for preventing and kill off disease, worm, grass etc.
The living body biological of evil biology, what biochemical pesticides referred to isolates from organism, with certain chemical constitution, to having
Evil biology has the bioactive substance of control action, which may be the metabolite of organism.
Microbial pesticide in biological pesticide is prevented harmful organism using biological living or its metabolite
A kind of preparation, be the material base and important means of biological control.Endophyte of plant (Endophyte) is certain phase or complete
Portion moves in fungi or bacterium inside the tissue and organ of health plant the stage.Endophyte of plant itself, which has from certain, to be driven
It is extracted in the plant of worm effect, it is good to pest controling effect, insect pest is solved from root, and plant life is not interfered in plant
It is long, free from environmental pollution, noresidue safe and non-toxic to people, animal, plant.Endophyte of plant kills high specificity to disease pest, while not
Natural enemy and beneficial organism are injured, ecological natural can be kept to balance.Raw materials for production and active ingredient belong to natural products, can return certainly
However reduce for naturally destroying, ensure sustainable development.At present have become potential microbial pesticide in biological control,
Yield increasing fungus is used as potential biological and ecological methods to prevent plant disease, pests, and erosion carrier bacterium.Endophyte generally existing in plant, quantity is very huge,
So far hundreds of kinds have been studied, potential quantity is even more to be difficult to estimate.Endophyte of plant as very important realm in microorganism,
Rich and varied biologically active natural products can be generated, and the control that its product is used for disease pest and weed is current micro- life
The important channel of object pesticide, fungicide jinggangmeisu and Polyoxin, insecticide avermectin and pleocidin etc. are at present extensively
It is general to come into operation.But the utilization of current endophyte of plant is mainly prevented or cure a disease aspect in agriculture degerming, and is killed in desinsection especially summer
Then rare report in terms of mosquito desinsection.
Invention content
The purpose of the present invention is to provide a kind of plant endogenesis bacteria pesticides and preparation method thereof.
For achieving the above object, technical scheme is as follows：
Plant endogenesis bacteria pesticide preparation method, including step：
S1：Endophyte of plant is extracted from Celastraceae plant；
S2：The endophyte of plant of purifying culture extraction is in case test；
S3：It is sprayed on drosophila and mosquito respectively after the metabolite of the endophyte of plant of culture is diluted, selects high cause
Dead rate plant endogenesis bacteria strain；
S4：The bacterial strain selected is subjected to fermented and cultured；
S5：It filters and the zymotic fluid for concentrating fermented and cultured obtains plant endogenesis bacteria pesticide.
Further, the step S1 is specifically included：
S11：The surface sterilization of vegetable material：Retain root soil when herborization, wraps up root with plastic cloth, keep it
Fresh and alive state；
S12：It takes the health tissues of the root of acquired plant, stem, leaf to be rinsed well with flowing water, is impregnated with 70% alcohol
3min, then 3min is impregnated with 2% sodium hypochlorite, finally with aseptic water washing 3 times removing the disinfection for being attached to material surface
Agent；
S13：100gl is taken to be coated on LB culture medium flat plates all over the sterile water embathed last, 28 DEG C are cultivated 72h hours
It is grown afterwards without microorganism, shows that surface sterilization is thorough；
S14：The separation of endophyte：Plant root, stem, leaf after the disinfection of face use sterile scissors longitudinal sectional respectively, are then placed in and go out
It is ground in bacterium mortar abundant；
S15：Buffer solution is added in plant tissue liquid after grinding and vibrates 1h under the conditions of 28 DEG C, l50rpm, is taken different dilute
The solution for releasing gradient is respectively coated on LB culture medium flat plates, 28 DEG C of constant temperature incubations 2-14 days, is selected after bacterium colony growth completely
Access LB agar slants preserve under the conditions of being placed in 4 DEG C；
S16：Colony morphological observation：There is scribing line on nitrogen culture medium to obtain single bacterium colony, observes single bacterium colony form and thalli morphology
And it records.
Further, the buffer solution is 10mlPBS buffer solutions.
Further, the step S3 is specifically included：It is sprayed respectively after the metabolite of the endophyte of plant of culture is diluted
It is sprinkled upon on drosophila and mosquito, selects high lethality plant endogenesis bacteria strain as strain to be tested；Strain to be tested is subjected to be commissioned to train more
It supports, is sprayed on drosophila and mosquito respectively after the dilution of the endophyte of plant metabolin after supporting that will more be commissioned to train, selects genetic stability
Good bacterial strain；The good bacterial strain of the genetic stability selected is continued to cultivate, and mouse is sprayed after its metabolite is diluted,
Using to the nontoxic bacterial strain of mouse as aimed strain.
Further, the step S5 is specifically included：The zymotic fluid for filtering and concentrating fermented and cultured, by the fermentation after concentration
Liquid is configured to pulvis, wettable powder, colloidal suspending agent, propellant or aerosol.
By the plant endogenesis bacteria pesticide obtained by above-mentioned preparation method.
Compared with prior art, beneficial effects of the present invention：
The plant endogenesis bacteria pesticide preparation method of the present invention, endophyte of plant screened itself, which has from certain, to be driven
It is extracted in the plant of worm effect, it is good to pest controling effect, insect pest is solved from root, and plant life is not interfered in plant
It is long, free from environmental pollution, noresidue safe and non-toxic to people, animal, plant；Endophyte of plant kills high specificity to disease pest, while not
Natural enemy and beneficial organism are injured, ecological natural can be kept to balance；Raw materials for production and active ingredient belong to natural products, can return certainly
However reduce for naturally destroying, ensure sustainable development；Since many factors and ingredient play a role, microorganism itself gathers around
Some genetic mutation, pest are difficult to develop immunity to drugs.
Specific implementation mode：
The extraction of 1 object endophyte of embodiment
The surface sterilization of vegetable material:Retain root soil when herborization, wraps up root with plastic cloth, keep its fresh and alive
State.It takes the health tissues of the root of acquired plant, stem, leaf to be rinsed well with flowing water, impregnates 3min with 70% alcohol, then use
2% sodium hypochlorite impregnates 3min, finally with aseptic water washing 3 times removing the disinfectant for being attached to material surface.It will be last
One time the sterile water embathed takes 100gl to be coated on LB culture medium flat plates, 28 DEG C culture 72h hours after grown without microorganism, table
Bright surface sterilization is thorough.
The separation of 2 endophyte of embodiment
Plant root, stem, leaf after the disinfection of face use sterile scissors longitudinal sectional respectively, are then placed in sterilizing mortar and grind fully.
In order to ensure that endogenetic bacteria is sufficiently separated, poured into the conical flask equipped with 10mlPBS buffer solutions after plant tissue grinding, 28
DEG C, vibrate 1h under the conditions of l50rpm, take 100g difference dilution gradients to be followed successively by 103、104、105With 106Juice be coated on LB training
It supports on base tablet, 28 DEG C of constant temperature incubations 2-14 days.After bacterium colony growth completely after select access LB agar slants be placed in 4 DEG C under the conditions of
It preserves.There is scribing line on nitrogen culture medium to obtain single bacterium colony, observation single bacterium colony edge shape, size, protuberance situation, configuration of surface, bacterium
Fall color, transparency and pigment production etc..After strain culturing 15h, Gram's staining is carried out, process is：1. strain is taken to train
Support object routine smear, drying, fixation；2. violet staining 1-2rain is added dropwise, washing：3. washing away residual water with iodine solution, iodine solution is used in combination
Cover about lmin, washing；4. residual water is sucked with filter paper, with dropper plus 95% ethanol decolorization, until when the ethyl alcohol of outflow is without purple,
It washes immediately：5. redying about 2min with sarranine, wash；6. after dry, Gram-stained yin and yang attribute and thalline are observed with oil mirror
Form, the length and width of thalline are surveyed with the micro- ruler of dressing table.
It is isolated from Celastraceae plant to 155 plants of endogenetic fungus.The isolated endogenetic fungus from Celastrus angulatus plant
Quantity is most, is 95 plants, and the endogenetic fungus being separated to from other 3 kinds of plants is less, respectively 25 plants, 18 plants and 12 plants.
In addition, there is also differences for quantity in plant different parts for plant endogenesis epiphyte.3 kinds of Celastrus angulatus, Euonymus japonicus and winter creeper are defended
The endophyte of lance section plant is distributed more, followed by stem in root, is finally fruit and other positions.
Embodiment 3 is tested and screening
Measure from 4 kinds of Celastraceae plants isolated 155 strain endophyte ferment filtrate insecticidal activity, with and mosquito
The drosophila for being all Diptera is that the insecticidal activity of 155 plants of endophytes of test worm pair carries out preliminary screening, the results showed that, by therein
8 kinds of endophyte metabolite dilutions, the rear death rate is 90% or more for 24 hours for drosophila, and the results are shown in Table 1.Pass through 5 plants of endophytes
Metabolite dilution, drosophila for 24 hours the death rate 70% or more.Mouse, mouse are sprayed after the metabolite of endophyte is diluted
It does not generate and significantly affects.The endophyte zymotic fluid and mycelia extract obtained to primary dcreening operation with 4 kinds of different color developing agents is shown
Whether colour response generates alkaloid compound with clear endophyte.The result shows that there is the intracellular generation of the endophyte of insecticidal activity
It thanks to object crude extract into positive reaction, tentatively concludes and wherein contain alkaloid compound.
1 endophyte ferment filtrate insecticidal activity assay result of table
| Test sample | The death rate (%) | Test sample | The death rate (%) |
| 1 | 30 | 8 | 10 |
| 2 | 60 | 9 | 47 |
| 3 | 20 | 10 | 65 |
| 4 | 55 | 11 | 5 |
| 5 | 10 | 12 | 16 |
| 6 | 30 | 13 | 13 |
| 7 | 69 | 14 | 24 |
4 plants of endophytes highest to lethality carry out genetic stability measurement, the results showed that：Produce the 4 of insecticide active substance
For strain endogenetic fungus when passage is to 5 generation, the tunning of two of which strain does not show culex pipiens pallens larvae desinsection work
Property, show that this two plants of bacterium may lose the ability for generating activated product, and other endophyte inheritance stabilities in cultured in vitro
Property measurement result show bacterial strain after passing on for 5 generations its generate Substance ability be not substantially reduced, show such side
Method can filter out less toxic efficient and good genetic stability endophyte of plant.
The preparation of 4 plant endogenesis bacteria pesticide of embodiment
The aimed strain filtered out is subjected to fermented and cultured, the zymotic fluid of fermented and cultured is filtered and concentrate, after concentration
Zymotic fluid is configured to pulvis, wettable powder, colloidal suspending agent, propellant or aerosol, can according to circumstances select to close in actual use
Suitable dosage form.
It should be understood that the application of the present invention is not limited to the above can for the normal technician of this field
With improvement or transformation based on the above description, all these modifications and variations should all belong to the guarantor of appended claims of the present invention
Protect range.
Claims (6)
1. plant endogenesis bacteria pesticide preparation method, which is characterized in that including step：
S1：Endophyte of plant is extracted from Celastraceae plant；
S2：The endophyte of plant of purifying culture extraction is in case test；
S3：It is sprayed on drosophila and mosquito respectively after the metabolite of the endophyte of plant of culture is diluted, selects high lethality
Plant endogenesis bacteria strain；
S4：The bacterial strain selected is subjected to fermented and cultured；
S5：It filters and the zymotic fluid for concentrating fermented and cultured obtains plant endogenesis bacteria pesticide.
2. plant endogenesis bacteria pesticide preparation method according to claim 1, which is characterized in that the step S1 is specifically wrapped
It includes：
S11：The surface sterilization of vegetable material：Retain root soil when herborization, wraps up root with plastic cloth, keep its fresh and alive
State；
S12：It takes the health tissues of the root of acquired plant, stem, leaf to be rinsed well with flowing water, impregnates 3min with 70% alcohol,
3min is impregnated with 2% sodium hypochlorite again, finally with aseptic water washing 3 times removing the disinfectant for being attached to material surface；
S13：100gl is taken to be coated on LB culture medium flat plates all over the sterile water that embathes last, nothing after 28 DEG C of cultures 72h hours
Microorganism grows, and shows that surface sterilization is thorough；
S14：The separation of endophyte：Plant root, stem, leaf after the disinfection of face use sterile scissors longitudinal sectional respectively, are then placed in sterilizing and grind
It is ground in alms bowl abundant；
S15：Buffer solution is added in plant tissue liquid after grinding and vibrates 1h under the conditions of 28 DEG C, l50rpm, takes different dilution ladders
The solution of degree is respectively coated on LB culture medium flat plates, 28 DEG C of constant temperature incubations 2-14 days, and access is selected after bacterium colony growth completely
LB agar slants preserve under the conditions of being placed in 4 DEG C；
S16：Colony morphological observation：There is on nitrogen culture medium scribing line obtain single bacterium colony, observation single bacterium colony form and thalli morphology are simultaneously remembered
Record.
3. plant endogenesis bacteria pesticide preparation method according to claim 2, which is characterized in that the buffer solution is
10mlPBS buffer solutions.
4. plant endogenesis bacteria pesticide preparation method according to claim 1, which is characterized in that the step S3 is specifically wrapped
It includes：It is sprayed on drosophila and mosquito respectively after the metabolite of the endophyte of plant of culture is diluted, selects high lethality plant
Endophyte bacterial strain is as strain to be tested；Strain to be tested is subjected to foster, the endophyte of plant metabolin that will more be commissioned to train after supporting of more being commissioned to train
It is sprayed on drosophila and mosquito respectively after dilution, selects the good bacterial strain of genetic stability；The genetic stability selected is good
Bacterial strain continue to cultivate, and will its metabolite dilution after spray mouse, using to the nontoxic bacterial strain of mouse as aimed strain.
5. plant endogenesis bacteria pesticide preparation method according to claim 1, which is characterized in that the step S5 is specifically wrapped
It includes：Zymotic fluid after concentration is configured to pulvis, wettable powder, colloidal suspending agent, spray by the zymotic fluid for filtering and concentrating fermented and cultured
Penetrate agent or aerosol.
6. by the plant endogenesis bacteria pesticide obtained by the preparation method described in any of the above claim.
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| CN201810620553.5A CN108739864A (en) | 2018-06-15 | 2018-06-15 | Plant endogenesis bacteria pesticide and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111011399A (en) * | 2019-11-01 | 2020-04-17 | 华侨大学 | Citrus psylla botanical insecticide and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111011399A (en) * | 2019-11-01 | 2020-04-17 | 华侨大学 | Citrus psylla botanical insecticide and preparation method thereof |
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Application publication date: 20181106 |