CN108651523A - Applications of the streptomycete NBF715 of one plant of separation in preparing Plant nematode insecticide - Google Patents
Applications of the streptomycete NBF715 of one plant of separation in preparing Plant nematode insecticide Download PDFInfo
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- CN108651523A CN108651523A CN201810408883.8A CN201810408883A CN108651523A CN 108651523 A CN108651523 A CN 108651523A CN 201810408883 A CN201810408883 A CN 201810408883A CN 108651523 A CN108651523 A CN 108651523A
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
-
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/22—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom rings with more than six members
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- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
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- Microbiology (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Applications of the streptomycete NBF715 detached the invention discloses one plant in preparing Plant nematode insecticide, applicant has found that streptomycete can be used for preventing various nematodes for the first time, compared with being prevented at present using chemical synthetic pesticide, since it comes from natural environment, have many advantages, such as efficient, less toxic, free from environmental pollution and be not likely to produce drug resistance, has broad application prospects.
Description
Technical field
The invention belongs to crops Prevention Technique fields, and in particular to the streptomycete NBF715 of one plant of separation is preparing plant
Application in nematode killer.
Background technology
Plant nematode endangers in the four big cause of diseases for causing plant infectious diseases is only second to fungi, be more than bacterium and
Virus.Root-knot nematode (Meloidogyne spp.) and cyst roundworm are important monoids therein.
Root-knot nematode worldwide occurs extensively, and host range is wide, often results in dropping in production over a large area for crop, gives
Agricultural production brings about great losses, according to estimation, about 50,000,000,000 dollars of the crop loss that the whole world thereby results in every year.Soybean born of the same parents
Cyst mematode is to seriously endanger the soil-borne disease of soybean yields and quality.The characteristics of disease occurs is that distribution is wide, weight of causing harm, passes
It is more to broadcast approach, general underproduction 10%-30%, serious plot is up to 70%-90%, or even total crop failure.
Chemical prevention is current prevention root knot nematode disease most efficient method, and according to its occupation mode, nematicide is main
It is divided into fumigant and non-fumigant.Fumigant needs to apply into the soil of certain depth, expands in gaseous form to upper and lower
It dissipates, because having stronger penetration power to soil, there is significant advantage in terms of forcing down First aggression insect population.But such medicament is mostly to go out
Natural disposition medicament seriously affects the beneficial organism in soil, if improper use, easily generates poisoning to working as stubble crop, and to dispenser skill
Art is more demanding.Non- fumigant pouring root use easily causes nematode drug resistance, while polluting environment and causing pesticide residue.Production
It is upper to be badly in need of safer effective ways.Biological control has obtained extensively as a kind of safely and effectively means in crop control of nematode
General application can improve product quality, and long-time service can also be played the role of improveing soil well, can for agriculture
Sustainable development provides safeguard, and exploitation new bio nematicide is of great significance.
Invention content
The streptomycete NBF715 detached the purpose of the present invention is to provide one plant is preparing the medicament for preventing or killing nematode
In application, the deposit number of the streptomycete NBF715 is CCTCC NO:M2017414.
It is also an object of the present invention to provide streptomycete NBF715 zymotic fluids active ingredients to prevent or kill preparing
The application gone out in the medicament of nematode, the streptomycete NBF715 zymotic fluid active ingredients are Boli mycin.
In order to achieve the above object, the present invention takes following technical scheme to realize：
The streptomycete NBF715 of one plant of separation is preparing the application in preventing or killing the medicament of nematode, including the use of this hair
Streptomycete NBF715 is prepared into nematode killer or control of nematode agent by bright conventional means, or with other auxiliary materials or effectively
Ingredient is prepared into seed treatment together, and the deposit number of the streptomycete NBF715 is CCTCC NO:M2017414.
In above-described application, it is preferred that be used to prepare into the seed treatment for preventing nematode；
In above-described application, it is preferred that when being used to prepare into seed treatment, including seed coat agent：
All components use sand mill milled processed after high-shear emulsion machine shearing.
In above-described application, it is preferred that when being used to prepare into seed treatment, including seed dressing：
All components are in sand mill ground and mixed.
In above-described application, it is preferred that the nematode includes root knot nematode, soy bean cyst roundworm.
Compared with prior art, the present invention has the following advantages：
(1) the present invention provides a kind of pale purple brown streptomycete, which is reported for the first time can be used for preventing crop nematodiasis,
With existing nematicide no interactions drug resistance.
(2) at present using chemical synthetic pesticide prevent compared with, since it comes from natural environment, have efficiently, low toxicity,
It is free from environmental pollution and the advantages that be not likely to produce drug resistance.
Specific implementation mode
In order to preferably explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
Present disclosure is not limited solely to following embodiment.Technical solution of the present invention is if not otherwise specified ability
The routine techniques in domain, the reagent or material derive from commercial channel if not otherwise specified.
Embodiment 1：
The acquisition and identification of pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715
The streptomycete of one plant of separation, from bestowing favour, Hefeng County soil is isolated, is identified through 16s rDNA combination Physiology and biochemistries
After belong to pale purple brown streptomycete (Streptomyces enissocaesilis), sent to Chinese Typical Representative on July 7th, 2017
Culture collection carries out preservation, Classification And Nomenclature：Pale purple brown streptomycete (Streptomyces enissocaesilis)
NBF715；Deposit number is CCTCC NO:M2017414；Place：China, Wuhan, Wuhan University.
In the present invention, it pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 or is called for short
NBF715。
Morphological feature：Bacterium colony is round, the smooth of the edge, surface compact.Aerial hyphae silver gray, substrate mycelium cork yellow,
Bacterium colony is round, non-pigment.Fibrillae of spores is distributed in straight-chain, spore oval or ellipse.
Bacterial strain physio-biochemical characteristics：
Bacterial strain NBF715 physio-biochemical characteristics-enzyme activity, carbon source oxidation
+：Positive reaction；-：Negative reaction；W：Weakly positive is reacted
Bacterial strain NBF715 physio-biochemical characteristics-produce acid using carbon source
+：The positive ,-：It is negative；w：Weakly positive
Embodiment 2：
The fermentation of pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715：
The preparation of pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed liquors：Including will be oblique
Face preserves strain and is activated in ISP-2 tablets, and picking is seeded in 100 milliliters of seed culture mediums, and at 28 DEG C, 160 revs/min are shaken
It is cultivated 4 days on bed；Up to seed liquor.
Seed culture based formulas is：1L culture mediums include 20g mannitol, 10g soy peptones, 0.35g biphosphates
Potassium, 0.5g calcium carbonate, 20 μ L of soya-bean oil, remaining is water.
Solid fermentation：Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed liquors are pressed
10% is connected in solid medium, sample-loading amount 20%, 30 DEG C culture 10d, the culture obtained in this way it is dense
Degree is 5*1010A spore/gram, as NBF715 microbial inoculums are used for following embodiment.
The solid fermentation culture medium is：83% wheat bran, 3% glucose, 5% potassium nitrate, 3% dipotassium hydrogen phosphate, 3%
Sodium chloride, 3% calcium carbonate, after the above formula mixing plus water stirs and evenly mixs, material-water ratio 10:6, the percentage is quality
Percentage.
Liquid fermentation：Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed liquors are pressed 5%
Amount is inoculated in fermentation medium, and temperature is 28 DEG C, 500 revs/min of the mixing speed of fermentation tank, and the loading amount of fermentation medium is tank
The 60% of body volume ferments 5 days, as NBF715 zymotic fluids, is used for following embodiment.
The formula of the fermentation medium includes：Mannitol 3%, soy peptone 3%, yeast powder 0.3%, calcium carbonate
0.5%, sodium chloride 0.5%；PH value 6.5-7.5, remaining is water, and the percentage is mass percent..
Embodiment 3：
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 prevents tomato root-knot eelworm
It tests and is carried out in tomato continuous cropping greenhouse in September, 2017, processing 1 is NBF715 bacterium of spreading manuer in holes when transplanting tomato
Agent, dosage are the caves 3g/, and subsequent earthing simultaneously pours root water.Processing 2 is filling NBF715 zymotic fluids, dosage 200mL/ after transplanting
, chemicals treatment is not done in control.4 cells are often handled, 3 weights are multiple, per cell 20m2.Normal farming management, 14 after dispenser
Week takes 15 plants of tomatoes at random in each cell, investigates plant disease grade.Grade scale：0 grade, 0-5% roots have root knot；1 grade, 6%-20
Root has root knot；2 grades, 21%-45% roots have root knot；3 grades, 46%-70% roots have root knot；；4 grades, 71%-85% roots have root knot；
5 grades, 86%-100% roots have root knot；：
Root knot index=∑ (plant number × rank at different levels)/(investigation total strain number × highest represents rank) × 100.
Control effect=(control root knot index-processing root knot index)/control root knot index × 100%
Table 1NBF715 microbial inoculums and zymotic fluid are to tomato root-knot eelworm control effect
Embodiment 4：
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 seed treatments prevent soybean cyst
Nematode
It tests and is carried out in successive soybean cropping field in May, 2017, processing 1 is seed dressing and soybean 1:30 (weight ratios) are dressed seed,
Processing 2 is suspension seed-coating agent 1:30 (weight ratios) carry out soybean coating, and control is not processed.Often handle 4 cells, 3 weights
It is multiple, per cell 20m2.Test the method for taking 5 repetitions of random district's groups.5 row areas of each processing, 5m rows are long, line-spacing 0.65m,
Plot area 16.25m2, 100 seeds of often row sowing.After soybean planting 50d, 25 young plants are chosen per cell, investigation single plant is big
Beans root system cyst raw amount, nematode population in stereomicroscope observation root is used in combination, and be converted into the quantity of every gram of root fresh weight nematode.
Cyst inhibiting rate (%)=(control cyst number-processing cyst number)/control cyst number × 100%；Nematode slip (%)=
(control line borer population-processing nematode number)/control line borer population × 100%.
The component and proportioning of the suspension seed-coating agent：
All components use sand mill milled processed after high-shear emulsion machine shearing to obtain the final product.
The component and proportioning of the seed dressing：
All components are in sand mill ground and mixed.
Table 2NBF715 suspension seed-coating agents and seed dressing influence soybean root system sporangiocyst and Gen Nei nematodes
| Processing | Cyst inhibiting rate (%) | Nematode slip (%) |
| Suspension seed-coating agent is coated | 73.78 | 83.29 |
| Seed dressing is dressed seed | 52.34 | 61.33 |
| Control | - | - |
Embodiment 4：
Pale purple brown streptomycete (Streptomyces enissocaesilis) NBF715 zymotic fluid active materials identification：
Zymotic fluid is transferred to and steel is not lured to be lyophilized in cup, sets in freeze dryer and freeze dried by NBF715 zymotic fluid 20L, is lyophilized
Condition is：Then cold-trap is cooled to -45 DEG C hereinafter, opening vacuum pump distillation by -40 DEG C of pre-freeze 2h in advance.Vacuum degree is 10-30Pa,
Heating rate is 2-3 DEG C/h, and maximum temperature-rise is 37 DEG C.After taking out freeze-dried powder, it is transferred in triangular flask, first with a small amount of 50% first
Alcohol-water moistening, adds 100mL ethyl acetate, in 180rpm oscillation extractions 1h on shaking table, detaches ester output phase, rotates, removes
Solvent, then dissolved with a small amount of methanol, it is aided with ultrasonic wave hydrotropy, is transferred in 1.5mL plastic centrifuge tubes, after centrifuging 2min, takes upper layer
Special tubule is transferred to be analyzed.
It can be seen that from uv absorption spectra, only there are one ultraviolet absorption peak, λ for the compoundmax=259nm.According to mass spectrum
The rule of figure can primarily determine from the mass spectrogram under negative ion mode first, and m/z489 is molecular ion peak, the one of the right
Place's higher peak is peak, this just illustrates the judgement of molecular ion peak.M/z982 under positive ion mode is [2M+H], is illustrated point
Daughter ion peak is m/z491, so that it is determined that the molecular weight of the compound is 490.According to the above UV absorption wavelength and molecular weight
Information, consult natural products database dictionary, the compound most to match be Boli mycin.
Embodiment 5：
Boli mycin eelworm-killing activity
Saprophitic nematode used in this experiment is Caenorhabditis elegans wild type N2, and nematode is cultivated on nematode growth media,
And using Escherichia coli OP50 as food, the synchronization of 20 DEG C of growth temperature, nematode is by the hermaphroditic adult to pregnancy
It carries out what alkaline lysis obtained, i.e., lysate is added in nematode suspension, being placed on sharp pounding 5min on trip misfortune oscillator makes to fill
Division solution.Ovum can resist cracking, and polypide is then dissolved in lysate.9 buffer solution rinsings of 5mL M are added in centrifuge tube to split
Liquid is solved, 3000rpm centrifuges 3min, removes supernatant, repeats this step 4 time.Gained ovum after rinsing is transferred to and is buffered containing M9
In the culture dish of liquid, 20 DEG C of culture 16-20h, ovum is the first-instar youngization hatched as synchronization, is used for toxotest.
About 300 Caenorhabditis elegans first-instar youngs are transferred to the culture dish of the 3.5cm of the ferment filtrate containing 1mL
In, it is control from blank PDB culture mediums.Chemical combination deka-gram object activity test methods are that Boli mycin is dissolved in dimethyl sulfoxide (DMSO)
(DMSO), it takes 5 μ L Boli mycin mother liquors to be dissolved in M9 buffer solutions, is configured to 1mL Boli mycin aqueous solutions, (eventually with dimethyl sulfoxide (DMSO)
It is a concentration of 0.5%) be control.Take nematode or the Meloidogyne incognita second instar larvae of about 300 Caenorhabditis elegans first-instar youngs
Addition contains 0,10 μ g/mL, 20 μ g/mL, and in the M9 buffer solutions of 40 μ g/mL Boli mycins, comparison medicament is lythidathion and fluorine
Pyrrole bacterium amide.20 DEG C of nematode culture casees are put in, the survival of nematode or The dead quantity are counted under rear microscope for 24 hours.It is raw
Survey the result shows that, Boli mycin to Caenorhabditis elegans have good insecticidal, with commodity medicament lythidathion and fluorine pyrrole bacterium acyl
Amine effect is suitable.
3 Boli mycin of table is to Caenorhabditis elegans insecticidal effect
| Medicament | Concentration | Preventive effect (%) |
| Boli mycin | 40μg/mL | 91.37 |
| Boli mycin | 20μg/mL | 73.56 |
| Boli mycin | 10μg/mL | 33.72 |
| Fosthiazate | 100μg/mL | 95.69 |
| 10μg/mL | 0 | |
| Fluopyram | 100μg/mL | 97.34 |
| 10μg/mL | 36.67 | |
| CK | - | - |
The tomato root for taking Meloidogyne incognita (greenhouse artificial culture and propagation) severe infections, surface is rinsed well with tap water
After silt, pieces of an egg are removed from root with tip tweezers, 0.5% (V/W) sodium hypochlorite is to pieces of an egg surface sterilization 2 times, every time
5min, then rinsed well repeatedly with sterile water.The pieces of an egg of disinfection are placed on the filter paper for suctioning water, 25 DEG C hatch 3-5 days；It incubates
The nematode dissolved can be used as bioassay.Bioassay carries out bioassay on 96 orifice plates, with reference to Masler's (2007)
Method.Per hole, sucking 190 μ l, 2 instar larvae nematode suspensions include nematode about 40,40 μ g/ml of Boli mycin are added, with 20 μ g/
Ml chloramphenicol inhibits varied bacteria growing.As a contrast with lythidathion.It acts on 3 days under the conditions of 8.0,25 DEG C of pH, is fallen in Olympus
Microscopically observation counting is set, counts per hole death borer population and calculates preventive effect.It is raw survey the result shows that, Boli mycin is to southern root
Tie lines worm has good insecticidal, suitable with commodity medicament lythidathion and fluopyram effect.
4 Boli mycin of table is to Meloidogyne incognita insecticidal effect
| Medicament | Concentration | Preventive effect (%) |
| Boli mycin | 40μg/mL | 88.18 |
| Fosthiazate | 40μg/mL | 83.26 |
Claims (5)
1. the streptomycete of one plant of separation is pale purple brown preparing the application in preventing or killing the medicament of nematode, the streptomycete
Streptomycete（Streptomyces enissocaesilis）NBF715；Deposit number is CCTCC NO: M2017414.
2. streptomycete fermentation liquid active ingredient is preparing the application in preventing or killing the medicament of nematode, the streptomycete fermentation
Liquid active ingredient is Boli mycin, and the streptomycete is pale purple brown streptomycete（Streptomyces enissocaesilis）
NBF715；Deposit number is CCTCC NO: M2017414.
3. application of the streptomycete of one plant of separation in preparing seed treatment, the streptomycete is pale purple brown streptomycete
（Streptomyces enissocaesilis）NBF715；Deposit number is CCTCC NO: M2017414.
4. application according to claim 3, the seed treatment：
Ingredients weight parts
Streptomycete fermentation liquid 80-95
Xanthans 1-4
PVA 1-10
Neopelex 0.5-2
Dispersant NNO 1-3；
The streptomycete fermentation liquid is that deposit number is CCTCC NO:The pale purple brown streptomycete of M2017414
（Streptomyces enissocaesilis）The liquid fermentation liquid of NBF715.
5. application according to claim 3, the seed treatment：
Ingredients weight parts
Drappus microbial inoculum 50-90
Xanthans 1-4
Neopelex 1-2
NNO 1-4
Filler 10-20；
The drappus microbial inoculum is that deposit number is CCTCC NO:The pale purple brown streptomycete of M2017414（Streptomyces enissocaesilis）NBF715 solid fermentation microbial inoculums.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789288A (en) * | 2021-11-03 | 2021-12-14 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Streptomyces JXGZ01, biological agent and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789288A (en) * | 2021-11-03 | 2021-12-14 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Streptomyces JXGZ01, biological agent and application |
| CN113789288B (en) * | 2021-11-03 | 2023-01-06 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Streptomyces JXGZ01, biological agent and application |
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