CN108651523B - Application of separated streptomyces NBF715 in preparation of plant nematode pesticide - Google Patents
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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
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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
- 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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Abstract
The invention discloses application of a separated streptomycete NBF715 strain in preparation of plant nematode insecticides, and the applicant finds that the streptomycete can be used for preventing and treating various nematodes for the first time.
Description
Technical Field
The invention belongs to the technical field of crop control, and particularly relates to application of a separated streptomyces NBF715 strain in preparation of plant nematode insecticides.
Background
Plant parasitic nematodes are second only to fungi, more than bacteria and viruses, in four main pathogens causing plant infectious diseases. Root-knot nematodes (melodogyne spp.) and cyst nematodes are among the important groups.
Root-knot nematodes occur widely worldwide, are widely hosted, often cause large-area production losses in crops, cause enormous losses to agricultural production, and are estimated to be lost in about $ 500 billion each year worldwide. Soybean cyst nematode disease is a soil-borne disease that seriously compromises soybean yield and quality. The disease is characterized by wide distribution, serious damage, multiple transmission ways, general yield reduction of 10-30 percent, severe land parcel up to 70-90 percent and even no yield.
Chemical control is currently the most effective method for controlling root knot nematode disease, and nematicides are largely divided into fumigants and non-fumigants, depending on their mode of use. The fumigant needs to be applied into soil with a certain depth, and diffuses upwards and downwards in a gas form, so that the fumigant has a strong penetrating power on the soil, and has a remarkable advantage in the aspect of reducing the initial infestation insect population. However, most of the pesticides are biocidal pesticides, which seriously affect beneficial organisms in soil, are liable to cause phytotoxicity to crops in the same crop if the pesticides are improperly used, and have high requirements on pesticide application technology. The non-fumigant root irrigation is easy to cause drug resistance of nematodes, and simultaneously pollutes the environment and causes pesticide residues. A safer and more effective method is urgently needed in production. Biological control is widely applied to crop nematode control as a safe and effective means, can improve the quality of products, can play a good role in improving soil after long-term use, provides guarantee for the sustainable development of agriculture, and has important significance in developing novel biological nematocides.
Disclosure of Invention
The invention aims to provide application of a separated streptomyces NBF715 strain in preparation of a medicament for preventing or killing nematodes, wherein the preservation number of the streptomyces NBF715 strain is CCTCC NO: M2017414.
The invention also aims to provide the application of the active ingredient of the streptomyces NBF715 fermentation liquor in preparing the medicament for preventing or killing the nematode, wherein the active ingredient of the streptomyces NBF715 fermentation liquor is the brigrecin.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the application of the separated streptomyces NBF715 in preparing the medicament for preventing or killing the nematodes comprises the step of preparing the streptomyces NBF715 into a nematode pesticide or a nematode control agent by using the conventional means of the invention, or preparing the streptomyces NBF715 into a seed treatment agent together with other auxiliary materials or active ingredients, wherein the preservation number of the streptomyces NBF715 is CCTCC NO: M2017414.
In the above-mentioned applications, preferably, the seed treatment agent is used for preparing a seed treatment agent for preventing nematodes;
in the above-mentioned applications, preferably, when used for preparing seed treatment agent, the seed treatment agent comprises:
all components are ground by a sand mill after being sheared by a high-shear emulsifying machine.
In the above-mentioned applications, it is preferable that, when used for preparing a seed treatment agent, the seed dressing agent comprises:
all the components are ground and mixed in a sand mill.
In the above application, preferably, the nematodes include vegetable root knot nematodes and soybean cyst nematodes.
Compared with the prior art, the invention has the following advantages:
(1) the invention provides a streptomyces lavendulae strain which is reported for the first time to be used for preventing and controlling crop nematode diseases and has no interactive drug resistance with the existing nematocides.
(2) Compared with the existing chemical synthetic pesticide, the pesticide has the advantages of high efficiency, low toxicity, no environmental pollution, difficult generation of drug resistance and the like because of being from natural environment.
Detailed Description
In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
obtaining and identifying Streptomyces enissocaisis NBF715
A separated Streptomyces is separated from soil in Enshi Hefeng county, belongs to Streptomyces lavendulae (Streptomyces enissocaiesiis) after being combined with 16s rDNA for physiological and biochemical identification, is delivered to a China center for type culture collection in 7 months and 7 days in 2017 for collection, and is classified and named: streptomyces lavendulae (Streptomyces enissocaisii) NBF 715; the preservation number is CCTCC NO of M2017414; a place: china, wuhan university.
In the present invention, Streptomyces enissocailis (NBF 715) or simply NBF715 is referred to.
Morphological characteristics: the colony is round, smooth in edge and compact in surface. Aerial hyphae are silvery gray, basal hyphae are cork yellow, colonies are round and pigment-free. The spore silk is distributed in straight chain shape, and the spore is oval or elliptical.
Physiological and biochemical characteristics of the strain:
bacterial strain NBF715 physiological and biochemical characteristics-enzyme activity and carbon source oxidation
+: positive reaction; -: negative reaction; w: weak positive reaction
Bacterial strain NBF715 physiological and biochemical characteristics-acid production by utilizing carbon source
+: positive, -: negative; w: weak positive
Example 2:
fermentation of Streptomyces lavendulae (Streptomyces enissocaisiis) NBF 715:
preparation of Streptomyces lavendulae (Streptomyces enissocaiesilis) NBF715 seed solution: comprises activating slant-stored strain on ISP-2 plate, selecting and inoculating to 100ml seed culture medium, and culturing on a shaking table at 28 deg.C and 160 rpm for 4 days; obtaining the seed liquid.
The formula of the seed culture medium is as follows: 1L of the culture medium comprises 20g of mannitol, 10g of soybean peptone, 0.35g of potassium dihydrogen phosphate, 0.5g of calcium carbonate, 20 mu L of soybean oil and the balance of water.
Solid fermentation: inoculating Streptomyces lavonoscens (Streptomyces enissocaisis) NBF715 seed solution 10% into solid culture medium, loading at 20%, and culturing at 30 deg.C for 10 days to obtain culture with concentration of 5 × 1010One spore/gram, NBF715 inoculum, was used in the examples below.
The solid fermentation medium comprises: 83 percent of bran, 3 percent of glucose, 5 percent of potassium nitrate, 3 percent of dipotassium hydrogen phosphate, 3 percent of sodium chloride and 3 percent of calcium carbonate, and the components are mixed and then added with water to be stirred and mixed uniformly, wherein the material-water ratio is 10:6, and the percentage is the mass percentage.
Liquid fermentation: the method comprises the following steps of inoculating 5% of Streptomyces enissocaisis (NBF 715) seed liquid into a fermentation medium at the temperature of 28 ℃, stirring at the speed of 500 rpm, wherein the loading amount of the fermentation medium is 60% of the volume of a tank body, and fermenting for 5 days to obtain NBF715 fermentation liquid which is used in the following examples.
The formula of the fermentation medium comprises: 3% of mannitol, 3% of soybean peptone, 0.3% of yeast powder, 0.5% of calcium carbonate and 0.5% of sodium chloride; the pH value is 6.5-7.5, the balance is water, and the percentage is mass percent. .
Example 3:
streptomyces lavendulae (Streptomyces enissocaiesiis) NBF715 for preventing and treating tomato root-knot nematode
The test is carried out in a tomato continuous cropping greenhouse in 2017, 9 months, wherein the treatment 1 is to apply NBF715 microbial inoculum in a hole mode with the dosage of 3 g/hole when the tomatoes are transplanted, and then soil is covered and rooting water is poured. The treatment 2 is implemented by filling NBF715 fermentation liquor after transplanting, the dosage is 200mL per plant, and the contrast is not treated by a medicament. Every 4 cells processed, 3 times repeated, 20m per cell2. And (4) carrying out normal farming management, randomly taking 15 tomatoes from each cell 14 weeks after application, and investigating the disease grade of the plants. GradingThe standard is as follows: 0 grade, 0-5% of roots have root knots; grade 1, 6-20 have root knots; grade 2, 21-45% of roots have root knots; grade 3, 46-70% of roots have root knots; (ii) a Grade 4, 71-85% of roots have root knots; grade 5, 86-100% of roots have root knots; :
the root knot index ∑ (number of plants at each stage × rank)/(total number of investigated plants × highest representative rank) × 100.
The control effect is (contrast root knot index-treatment root knot index)/contrast root knot index multiplied by 100 percent
TABLE 1NBF715 strains and fermentation broths for control of tomato root-knot nematodes
Example 4:
streptomyces lavendulae (Streptomyces enissocaisis) NBF715 seed treatment agent for preventing and treating soybean cyst nematode
The test was carried out in the continuous cropping soybean field in 5 months of 2017, with treatment 1 being seed dressing agent and soybean 1:30 (weight ratio) seed dressing, treatment 2 being suspension seed dressing agent 1:30 (weight ratio) soybean coating, and the control being untreated. Every 4 cells processed, 3 repetitions, 20m per cell2. The test was performed by 5 replicates in a randomized block. Each treatment line has 5 lines of area, 5m line length, 0.65m line spacing and 16.25m cell area2100 seeds were sown in each row. After the soybeans are sowed for 50 days, 25 seedlings are selected in each cell, the cyst occupancy of the root system of a single soybean plant is investigated, the number of nematodes in the root is observed by a stereoscopic microscope, and the number of the nematodes is reduced to the number of fresh weight nematodes per gram of the root. Cyst inhibition (%) - (control cyst number-treated cyst number)/control cyst number × 100%; the nematode reduction (%) was (control nematode count-treatment nematode count)/control nematode count × 100%.
The suspension seed coating agent comprises the following components in percentage by weight:
all the components are sheared by a high-shear emulsifying machine and then ground by a sand mill to obtain the high-performance emulsion.
The seed dressing agent comprises the following components in percentage by weight:
all the components are ground and mixed in a sand mill.
TABLE 2NBF715 suspended seed coating and seed dressing effect on soybean root cysts and intraradicular nematodes
| Treatment of | Cyst inhibition (%) | Nematode reduction (%) |
| Suspended seed coating agent coating | 73.78 | 83.29 |
| Seed dressing agent for dressing seeds | 52.34 | 61.33 |
| Control | - | - |
Example 4:
active substance identification of Streptomyces lavendulae (Streptomyces enissocaiesiis) NBF715 fermentation liquor:
and (3) 20L of NBF715 fermentation liquor, transferring the fermentation liquor into a non-induced steel freeze-drying cup, and freeze-drying in a freeze dryer under the freeze-drying conditions: pre-freezing at-40 deg.C for 2h, pre-cooling the cold trap to-45 deg.C, and starting vacuum pump to sublimate. The vacuum degree is 10-30Pa, the heating rate is 2-3 ℃/h, and the limit temperature rise is 37 ℃. Taking out the freeze-dried powder, transferring to a triangular flask, wetting with a small amount of 50% methanol-water, adding 100mL ethyl acetate, performing oscillation extraction for 1h at 180rpm on a shaking table, separating an ester phase, performing rotary evaporation, removing a solvent, dissolving with a small amount of methanol, assisting in ultrasonic dissolution, transferring into a 1.5mL plastic centrifugal tube, centrifuging for 2min, and transferring the upper layer into a special small tube for analysis.
As can be seen from the ultraviolet absorption spectrogram, the compound only has one ultraviolet absorption peak, lambdamax259 nm. According to the rule of mass spectrograms, firstly, the mass spectrogram in a negative ion mode can be preliminarily determined, m/z489 is a molecular ion peak, and the higher peak on the right side is a peak, which just indicates the judgment of the molecular ion peak. M/z982 in positive ion mode is [2M + H]The molecular weight of this compound was determined to be 490 by specifying the molecular ion peak as m/z 491. According to the information of the ultraviolet absorption wavelength and the molecular weight, a natural product database dictionary is consulted, and the most matched compound is the brigrecin.
Example 5:
brogliomycin nematicidal activity
The saprophytic nematodes used in the experiment are caenorhabditis elegans wild type N2, the nematodes are cultured on a nematode growth medium, escherichia coli OP50 is used as food, the growth temperature is 20 ℃, the synchronization of the nematodes is obtained by carrying out alkaline cracking on pregnant hermaphrodite adults, namely, a lysate is added into a nematode suspension, and the nematode suspension is placed on a free-running oscillator to strongly shake for 5min so as to fully crack. Eggs resist lysis, while worm bodies dissolve in the lysis solution. Add 5mL M9 buffer to rinse the lysate into the centrifuge tube, centrifuge at 3000rpm for 3min, remove the supernatant, and repeat this step 4 times. Transferring the rinsed eggs to a culture dish containing M9 buffer solution, and culturing at 20 ℃ for 16-20h to obtain eggs which are hatched into synchronous first-instar larvae for toxicity test.
Approximately 300 caenorhabditis elegans first instar larvae were transferred to a 3.5cm petri dish containing 1mL of fermentation filtrate, from blank PDB medium as a control. Compound activity was tested by dissolving brivarycin in dimethyl sulfoxide (DMSO), taking 5. mu.L of brivarycin mother solution and dissolving in M9 buffer to prepare 1mL of brivarycin aqueous solution, using dimethyl sulfoxide (final concentration of 0.5%) as control. About 300 first instar larvae of caenorhabditis elegans or second instar larvae of Meloidogyne incognita were added to M9 buffer containing 0, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL brivaramycin and the control agents were fosthiazate and fluopyram. Placing in a nematode incubator at 20 ℃, and counting the survival or death number of nematodes under a microscope after 24 hours. Bioassay results show that the brix has good insecticidal activity on caenorhabditis elegans, and has the same effect as commercial medicaments of fosthiazate and fluopyram.
TABLE 3 pesticidal effect of bleomycin on caenorhabditis elegans
| Medicament | Concentration of | Control effect (%) |
| Brogliomycin | 40μg/mL | 91.37 |
| Brogliomycin | 20μg/mL | 73.56 |
| Brogliomycin | 10μg/mL | 33.72 |
| Thiazolylphosphines | 100μg/mL | 95.69 |
| 10μg/mL | 0 | |
| Fluopyram | 100μg/mL | 97.34 |
| 10μg/mL | 36.67 | |
| CK | - | - |
Collecting tomato root seriously infected by Meloidogyne incognita (greenhouse artificial culture and propagation), washing with tap water to remove silt on surface, taking off ovum block from root with sharp-pointed tweezers, sterilizing ovum block surface with 0.5% (V/W) sodium hypochlorite for 5min for 2 times, and repeatedly washing with sterile water. Placing the sterilized egg mass on a filter paper which is sufficiently absorbed with water, and incubating for 3-5 days at 25 ℃; the hatched nematodes can be used as a bioassay. Bioassay was performed in 96-well plates, according to the method of Masler (2007). Mu.l of 2-instar nematode suspension containing about 40 heads of nematodes was aspirated into each well, 40. mu.g/ml of brithromycin was added, and growth of the heteromycete was inhibited with 20. mu.g/ml of chloramphenicol. Fosthiazate was used as a control. The mixture is acted for 3 days under the conditions of pH 8.0 and 25 ℃, observed and counted under an Orlinbas inverted microscope, the number of dead insects in each hole is counted, and the control effect is calculated. Bioassay results show that the brigrecin has good insecticidal activity on southern root knot worms and has the same effect as commercial medicaments of fosthiazate and fluopyram.
TABLE 4 insecticidal Effect of Blulicin on Meloidogyne incognita
| Medicament | Concentration of | Control effect (%) |
| Brogliomycin | 40μg/mL | 88.18 |
| Thiazolylphosphines | 40μg/mL | 83.26 |
Claims (5)
1. The application of the separated Streptomyces in preparing the medicament for preventing or killing the nematodes is that the Streptomyces is Streptomyces enissocaisii NBF 715; the preservation number is CCTCC NO: M2017414, and the nematodes are soybean cyst nematodes, tomato root knot nematodes and caenorhabditis elegans wild type N2.
2. The application of the effective component of the Streptomyces fermentation liquor in preparing the medicament for preventing or killing the nematodes, wherein the effective component of the Streptomyces fermentation liquor is the brivard, and the Streptomyces is Streptomyces enissocaisis (NBF 715); the preservation number is CCTCC NO: M2017414, and the nematodes are soybean cyst nematodes, tomato root knot nematodes and caenorhabditis elegans wild type N2.
3. The application of the separated Streptomyces in preparing the seed treating agent is that the Streptomyces enissocaisis (NBF 715) is selected; the preservation number is CCTCC NO: M2017414.
4. The use according to claim 3, wherein the seed treatment agent:
the Streptomyces fermentation liquid is liquid fermentation liquid of Streptomyces lavonoensis (Streptomyces enissocaisii) NBF715 with the preservation number of CCTCC NO: M2017414.
5. The use according to claim 3, wherein the seed treatment agent:
the streptomyces inoculant is a Streptomyces lavendulae (Streptomyces enissocaiensis) NBF715 solid fermentation inoculant with the preservation number of CCTCC NO: M2017414.
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