WO2023116710A1 - 高产多杀菌素菌株及其用途 - Google Patents

高产多杀菌素菌株及其用途 Download PDF

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WO2023116710A1
WO2023116710A1 PCT/CN2022/140418 CN2022140418W WO2023116710A1 WO 2023116710 A1 WO2023116710 A1 WO 2023116710A1 CN 2022140418 W CN2022140418 W CN 2022140418W WO 2023116710 A1 WO2023116710 A1 WO 2023116710A1
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mass
spinosyn
parts
compounds
water
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PCT/CN2022/140418
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English (en)
French (fr)
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郭强
曹海峰
靳志刚
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山东鲁抗医药股份有限公司
山东鲁抗生物农药有限责任公司
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Publication of WO2023116710A1 publication Critical patent/WO2023116710A1/zh

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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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P5/00—Nematocides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/02—Acaricides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure belongs to the technical field of biological fermentation, in particular, the disclosure relates to a high-yield spinosad strain and its application.
  • Pesticides As an indispensable means of production in agricultural production, pesticides have made great contributions to agricultural development and human food supply. Pesticides mainly include three categories: fungicides, insecticides and herbicides. There are thousands of formulated insecticide products with chemicals as active ingredients in the world. The aforementioned insecticide products include antibacterials, acaricides, fungicides, nematocides, animal baths, avicides and disinfectants. Chemical control has the advantages of quick effect, remarkable effect, convenient use, and is not restricted by regions and seasons. It is still the main means of pest control for large-scale control.
  • pesticides For the human body, pesticides often cause damage through the skin, respiratory system and digestive system, which can bring about acute toxic reactions to pesticides such as poisoning, skin and eye irritation, and possible long-term effects, such as causing cells in the body to produce Gene mutation, change genetic cell mechanism, cause cancer or deformity, etc.
  • spinosad is a low toxicity insecticide.
  • Spinosad is a broad-spectrum insecticide, which can effectively control pests of Lepidoptera, Diptera and Thysanoptera, and can well control certain pest species in Coleoptera and Orthoptera that devour leaves in large quantities. It is relatively low toxicity to mammals and birds, and only slightly to moderately toxic to aquatic animals. Considering the above factors, spinosad is usually the first choice when carrying out integrated pest management.
  • the purpose of the present disclosure is to provide a new high-yielding spinosyn strain, which can produce a variety of spinosyns with high yields, wherein spinosyn A and spinosyn D are The total output of shake flask is about 6g/L.
  • the present disclosure provides a high-yield spinosad strain, which is a soil actinomycete of the genus Saccharopolyspora, namely Saccharopolyspora spinosa (Saccharopolyspora spinosa) W618, and its preservation number is CCTCC NO: M 20211261.
  • the present disclosure provides a pesticidal composition comprising the strain.
  • the composition further comprises one or more compounds selected from the group consisting of organophosphorus compounds, carbamate compounds, pyrethroid compounds, acylurea compounds, other types of insect growth regulators, and Insect hormone analogues, neonicotinoids and other nicotinic compounds, macrolide compounds, other insecticidal, acaricidal, molluscicidal and nematicidal compounds or active ingredients and phagocytosis stimulators.
  • organophosphorus compounds carbamate compounds, pyrethroid compounds, acylurea compounds, other types of insect growth regulators, and Insect hormone analogues, neonicotinoids and other nicotinic compounds, macrolide compounds, other insecticidal, acaricidal, molluscicidal and nematicidal compounds or active ingredients and phagocytosis stimulators.
  • the pesticidal composition may further comprise a pharmaceutically acceptable carrier, such as a liquid carrier or a solid carrier,
  • the liquid carrier may be selected from aliphatic ketones (such as cyclohexanone) and complex alcohols (such as 2-ethoxyethanol) and aromatic compounds, especially xylene and petroleum fractions, especially petroleum One or more of the high-boiling naphthalene and olefin moieties such as heavy aromatic naphtha.
  • aliphatic ketones such as cyclohexanone
  • complex alcohols such as 2-ethoxyethanol
  • aromatic compounds especially xylene and petroleum fractions, especially petroleum
  • high-boiling naphthalene and olefin moieties such as heavy aromatic naphtha.
  • the solid support may be selected from one or more of attapulgite clay, montmorillonite clay, diatomaceous earth, or purified silicate.
  • the dosage form of the pesticidal composition can be selected from wettable powder, soluble powder, emulsifiable concentrate, water suspension, dispersible oil suspension, water emulsion, aerosol, microemulsion and water dispersible granule .
  • the present disclosure provides a preparation method of the aforementioned pesticidal composition, the preparation method comprising inoculating the aforementioned bacterial strain into a medium for fermentation and culturing to prepare the pesticidal composition.
  • the medium includes seed medium and fermentation medium.
  • the seed culture medium and/or fermentation medium is adjusted to pH with a pH regulator; preferably, the pH regulator is an alkali; more preferably, the alkali is sodium hydroxide, potassium hydroxide, carbonic acid One or more of sodium and sodium bicarbonate.
  • the pH regulator is an alkali; more preferably, the alkali is sodium hydroxide, potassium hydroxide, carbonic acid One or more of sodium and sodium bicarbonate.
  • the fermentation conditions are 200-300rpm, 10-40°C, and 50-70% humidity;
  • the fermentation conditions are 250rpm, 28°C, and 60% humidity.
  • the present disclosure provides the use of the aforementioned insecticidal composition in the preparation of drugs for killing insects, killing acaria, and killing nematodes.
  • the insecticidal, acaricidal, and nematicidal drugs can be applied to animals related to agriculture, such as cattle, pigs, sheep, goats, horses or donkeys, or plants, such as soybeans, Corn, sugar cane, canola, potato, cotton, rice, coffee, citrus, almonds, tea.
  • the medicament may be enterally administered to animals, such as in the form of tablets, capsules, drinks, drenches, granules, pastes, boluses, and suppositories.
  • the drug is administered parenterally to an animal, such as by injection (including intramuscular, subcutaneous, intravenous, intraperitoneal, etc.), implantation, showering, dipping, spraying, pouring, dipping, washing , the drug for application in the form of a dusting powder.
  • the drug is administered to plants by spraying, pouring, dripping, washing, or dusting.
  • Saccharopolyspora spinosa (Saccharopolyspora spinosa) W618 was deposited in the China Center for Type Culture Collection (CCTCC for short, the address is Wuhan University, Wuhan, China) on October 27, 2021, and the preservation number is CCTCC NO: M 20211261.
  • the pesticidal composition containing Saccharopolyspora spinosa W618 and/or its fermentation product and/or its bacterial suspension and/or its culture solution can effectively control the pests of Lepidoptera, Diptera and Thysanoptera, and can Excellent control of certain species of leaf-eating pests of Coleoptera and Orthoptera.
  • Figure 1 shows the results of detecting the components of the fermented product by LC-MS, wherein the upper figure is the result of detecting the fermented product in Example 1, and the lower figure is the detection result of the standard spinosad.
  • Figure 2 shows the results of using LC to detect various spinosyns in the fermented product.
  • spinosyn A and spinosyn D spinosyn A-iso and spinosyn B can also be detected in the fermented product , Spinosyn E, Spinosyn H, Spinosyn J, Spinosyn K, Spinosyn L, Spinosyn L2, Spinosyn L3, Spinosyn P, Spinosyn P-CH 2 .
  • Fig. 3 shows the yield results after screening 20 strains of S. spinosa strain W600 after 6 rounds of mutagenesis and fermenting one by one, wherein the S. spinosa strain with the highest yield is W618.
  • Fig. 4 shows the photographs of mature colony cultured from seeds of S. spinosa strain W600.
  • fertilization product refers to a product produced by a process that includes a fermentation step using a fermenting organism.
  • bacterial suspension refers to the mixture obtained by solid-liquid separation of the fermented liquid after the fermentation of the fermenting organisms is completed, and is usually separated and collected by centrifugation or membrane filtration.
  • broth refers to a liquid culture containing both the fermenting organism and the culture medium.
  • Plant in this disclosure includes seedlings, shrubs and trees. Plants are to be understood as also including plants which have been rendered tolerant to herbicides or classes of herbicides (for example ALS-, GS-, EPSPS-, PPO- and HPPD-inhibitors) by conventional breeding or by genetic engineering methods receptive crops. Plants are also to be understood as being obtained by conventional methods of breeding or genetic engineering and comprising so-called output traits (eg improved storage capacity, higher nutritional value and improved aroma).
  • herbicides or classes of herbicides for example ALS-, GS-, EPSPS-, PPO- and HPPD-inhibitors
  • insecticidal composition in the present disclosure refers to a composition having an insecticidal effect on pests.
  • control means that the pesticidal composition is effective to reduce the incidence of the corresponding parasite to a harmless level in an animal infected with such a parasite. More specifically, as used herein, “control” means that the active compounds are effective in killing the corresponding parasite, inhibiting its growth or inhibiting its reproduction.
  • the pesticidal compositions according to the invention can be applied directly.
  • these pesticidal compositions may contain pharmaceutically acceptable carriers and/or adjuvants known in the art.
  • arthropod pests that may be controlled by the "pesticidal compositions" described in the present disclosure include the following: Amblyomma americanum, Amblyomma maculatum, Argas persicus, Boophilus microplus, Dermacentor anderson, Dermacentor variabilis, Dermanyssus gallinae, Ixodes ricinus, Rhipicephalus sanguineus, Itch mite (Chorioptes spp.), Bovine mite (Demodex bovis), Dog mite (Demodex canis), Chicken bump mite (Knemidokoptes gallinae), Fowl bump mite (Knemidokoptes mutans), Otobius megnini (ear tick), Psoroptes equi, Psoroptes ovis, Sarcoptes scabiei, Aedes, Anopheles, Culex, Pleurotus ( Culiseta), Bovicola bovis(cattle biting louse), Callitrog
  • the pesticidal composition provided by the present disclosure can be used to control endoparasites and ectoparasites. These pesticidal compositions are administered in a known manner (by enteral administration in the form of, for example, tablets, capsules, drinks, drenches, granules, pastes, boluses, suppositories; by parenteral administration to animals or plant administration).
  • Drugs such as by injection (including intramuscular, subcutaneous, intravenous, intraperitoneal, etc.), implantation, by taking, for example, showering or dipping, spraying, pouring and dipping, washing, dusting and in the form of insecticidal Application (e.g., administration) of the composition with the aid of shaped articles (e.g., roller rings, ear tags, tail tags, limb bands, reins, manufacturing devices, and the like).
  • injection including intramuscular, subcutaneous, intravenous, intraperitoneal, etc.
  • implantation by taking, for example, showering or dipping, spraying, pouring and dipping, washing, dusting and in the form of insecticidal Application (e.g., administration) of the composition with the aid of shaped articles (e.g., roller rings, ear tags, tail tags, limb bands, reins, manufacturing devices, and the like).
  • shaped articles e.g., roller rings, ear tags, tail tags, limb bands, reins, manufacturing devices, and
  • the yield of spinosyn compounds produced by using S. spinosa is relatively low, resulting in high cost for industrial preparation of spinosyn compounds.
  • the inventor obtained a spinosad high-yielding strain Saccharopolyspora spinosa (Saccharopolyspora spinosa) W618 from a large number of strains through conditional screening of spinosyn production and multiple rounds of ultraviolet mutagenesis, and used its fermentation to prepare spinosad shake flasks Yield up to 6g/L. Therefore, S. spinosa W618 can be developed as an industrial strain with high spinosad production.
  • the present disclosure provides a high-yield spinosad strain, which is a soil actinomycete of the genus Saccharopolyspora, namely Saccharopolyspora spinosa (Saccharopolyspora spinosa) W618, and its preservation number is CCTCC NO: M 20211261.
  • the present disclosure provides a pesticidal composition comprising the aforementioned S. spinosa.
  • the pesticidal composition comprises spinosyn A, spinosyn A-iso, spinosyn B, spinosyn C, spinosyn D, spinosyn E, spinosyn F, Spinosyn G, Spinosyn H, Spinosyn J, Spinosyn K, Spinosyn L, Spinosyn L2, Spinosyn L3, Spinosyn P, Spinosyn P-CH 2 and more than 30 One or more of the structurally similar components.
  • the pesticidal composition comprises spinosyn A and spinosyn D.
  • the pesticidal composition further comprises one or more compounds selected from the group consisting of organophosphorus compounds, carbamate compounds, synthetic pyrethroid compounds, acylurea compounds, other types of insect growth regulators insecticides and insect hormone analogs, neonicotinoids and other nicotinic compounds, macrolide compounds, other insecticidal, acaricidal, molluscicidal and nematicidal compounds or active ingredients and phagocytosis stimulators.
  • the organophosphorus compound is selected from one or more of the following compounds: aceton, azinphos-phos, thiamephos, chlorphos, chlorpyrifos, phosphinophos, dematon, systemic Phosphorus, diazinon, dichlorvos, dimethoate, EPN, erthoate, methoxaphos, oxypyrimiphos, grammefos, fenitrothion, fonsofos, fenthion, difenfos, ango, thiazolonephos, Aphidfos (heptapenphos), malathion, methamidophos, methyl parathion (methyl 1605), amephos, monocrotophos, parathion (1605), Phorate, phosalon, imophos, phosphamide, etprofocarb, oxafos (cyanoxime),
  • the carbamate compound is selected from one or more of the following compounds: aldicarb, bentacarb, carbofuran, dimesulfon, BPMC, oxybufocarb ( butoxycarbocim), Carbaryl (Carbaryl), Carbocarb (Carbocicarb), Carbosulfan Carbosulfan, Dixiancarb, Benchoncarb, Butylcarbocim, Fuxincarb, Meethecarb, Isoprocarb, Methocarb, methoxacarb, pirimicarb, monsoxur, propoxur, thiodicarb, and thiofurox.
  • the synthetic pyrethrin compound is selected from one or more of the following compounds: bifluethrin, allethrin, ⁇ -cyfluthrin, bifenthrin, dextropyrethrin Esters, cyfluthrin, Glinneid, ⁇ -Greinne, ⁇ -Greneline, cypermethrin, ⁇ -cypermethrin, ⁇ -cypermethrin, deltamethrin, esfenvalerate, permethrin, penfluthrin, Fenpropathrin, Flucyfluthrin, Flufluthrin, Chlorfluvalinate, Tau-Chlorfluvalinate, Hhalofenthrin, Permethrin, Protrifenbute, Pyrethrin, Pyrethrin, Tefluthrin, Tefluthrin, Tefluthrin, Fish Safe Pyrethrins such as Etofenprox, Natural
  • the acylureas, other types of insect growth regulators and insect hormone analogs are selected from one or more of the following compounds: buprofezin, chromfenozide, difenoxuron, verflubenzuron (flubenzuron) urea kill), fenoxycarb, fluflubenzuron, terbufenazide, hexaflumuron, Mengwuyier, flupropoxycarbamide, Mengwuyiwu, methoxyfenozide, diflufenuron, mosquitoes Ether, pyrifluron and bisphenylhydrazide, N-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]- N'-(2,6-difluorobenzoyl)urea.
  • the neonicotinoids and other nicotinoids are selected from one or more of the following compounds: acetamiprid, AKD-1022, badan, TI-435, clothhiamidin, MTI-446, dinotefuran, imidacloprid, nicotine, nitrapyridine, thiamethoxam, thiacloprid.
  • the macrolide compound is selected from one or more of the following compounds: avermectin, milbemycin, abamectin, ivermectin, milbemycin and emamectin benzoate.
  • the other insecticidal, acaricidal, molluscicidal and nematicidal compounds or active ingredients are selected from one or more of the following compounds: , Cyclotin, Bifenazate, Bromofen, Chlorbenzamidine, Chlorfenapyr, Chlofentezine, Ethyl Fofol, Chlordane, Tricyclotin, Cyromazine, DDT, Dicofol, Dildrin , DNOC, endosulfan, ethoxazole, fenazaquin, fenzafen, fenpyroximate, ⁇ -pyroxafen, fipronil, flubenimide thiazole, hexymethoxazole, IKI-220, oxadiazole, lindane , methoxycarb, snail enemy, methoxyDDT, neem, petroleum and vegetable oil, pyridaben
  • the phagocytosis stimulating agent is selected from one or more of the following compounds: cucurbitacins, sugars and Coax.
  • the pesticidal composition may further comprise a pharmaceutically acceptable carrier, such as a liquid carrier, or a solid carrier.
  • a pharmaceutically acceptable carrier such as a liquid carrier, or a solid carrier.
  • the liquid carrier can be selected from aliphatic ketones (such as cyclohexanone) and complex alcohols (such as 2-ethoxyethanol) and aromatic compounds, especially xylene and petroleum fractions, especially high-boiling naphthalenes of petroleum and one or more of olefinic moieties such as heavy aromatic naphtha.
  • aliphatic ketones such as cyclohexanone
  • complex alcohols such as 2-ethoxyethanol
  • aromatic compounds especially xylene and petroleum fractions, especially high-boiling naphthalenes of petroleum and one or more of olefinic moieties such as heavy aromatic naphtha.
  • the solid carrier can be selected from one or more of attapulgite clay, montmorillonite clay, diatomaceous earth or purified silicate.
  • the dosage form of the pesticidal composition is selected from wettable powder, soluble powder, emulsifiable concentrate, water suspension, dispersible oil suspension, water emulsion, aerosol, microemulsion and water dispersible granules agent.
  • the pesticidal compositions described herein may be provided together with pharmaceutically acceptable carriers to provide, for example, baits, concentrated emulsions, powders, emulsified concentrates, fumigants, gels, granules, microcapsules, seed treatments formulations, suspension concentrates, suspoemulsions, tablets, water-soluble liquids, water-dispersible granules or dry flowable wettable powders and ultra-low volume solutions.
  • pharmaceutically acceptable carriers for example, baits, concentrated emulsions, powders, emulsified concentrates, fumigants, gels, granules, microcapsules, seed treatments formulations, suspension concentrates, suspoemulsions, tablets, water-soluble liquids, water-dispersible granules or dry flowable wettable powders and ultra-low volume solutions.
  • Aqueous suspensions or emulsions of pesticidal compositions can frequently be used.
  • Such water-soluble, water-suspendable or emulsifiable formulations are solids (often known as wettable powders or water-dispersible granules) or liquids (often known as emulsifying concentrates, or aqueous suspensions).
  • Wettable powders which can be compacted to form water-dispersible granules, comprise an intimate mixture of the pesticidal composition, carrier and surfactant.
  • the support is usually selected from attapulgite clays, montmorillonite clays, diatomaceous earth or purified silicates.
  • Effective surfactants are selected from sulfonated lignins, fused naphthalene sulfonates, naphthalene sulfonates, alkylbenzene sulfonates, alkyl sulfonates and nonionic surfactants such as ethylene oxide adducts of alkylphenols.
  • Emulsifiable concentrates of the claimed pesticidal compositions are generally from about 50 to about 500 grams of composition per liter of liquid, equal to about 10% to about 50%, dissolved in an inert carrier which is a water-immiscible solvent Mixture with emulsifier.
  • Organic solvents include organics, such as xylenes, and petroleum fractions, such as the high boiling naphthalenic and olefinic portions of petroleum, including heavy and aromatic naphthas. Other organics such as terpene solvent-rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols can also be used.
  • Emulsifiers for emulsifiable concentrates are typically mixed ionic and/or nonionic surfactants, such as those mentioned herein or their equivalents.
  • Aqueous suspensions can be prepared to contain the water-insoluble compounds of the pesticidal compositions of this invention, wherein the compounds are dispersed in an aqueous vehicle at a concentration generally in the range of about 5 to about 50% by weight.
  • Suspensions are prepared by finely grinding the pesticidal composition, vigorously mixing in an excipient of water, surfactants and dispersing agents.
  • Inert ingredients such as inorganic salts and synthetic or natural gums, can also be employed, as desired, to increase the density and/or viscosity of the aqueous vehicle.
  • Precipitating fluids can be prepared by dissolving the active molecule in a water-miscible solvent and a surfactant or surface-active polymer. On mixing these formulations with water, the active compound precipitates out and the surfactant controls the size of the resulting microcrystalline precipitate. Crystal size can be controlled by selecting a specific polymer and surfactant mixture.
  • the pesticidal composition can also be prepared as granules and applied to the soil.
  • the pesticidal composition is dispersed in an inert carrier, usually clay or equivalent.
  • granules are prepared by dissolving the pesticidal composition in a suitable solvent and applying it to a granular carrier which has been processed to the desired particle size.
  • the particle size is typically between about 0.5mm and 3mm.
  • Granules can also be prepared by forming a dough or paste of the carrier and the pesticidal composition, drying the mixture, and comminuting the dough or paste to the desired particle size.
  • pesticidal compositions can also be mixed with suitable organic solvents.
  • Organic solvents are generally mild solvents widely used in agriculture. These combinations are usually given as a spray. More commonly, pesticidal compositions are applied as dispersions in a liquid carrier, where the liquid carrier is water.
  • the pesticidal composition can also be applied as an aerosol composition.
  • the pesticidal composition is dissolved in an inert carrier which is a pressure-generating propellant mixture. Aerosol compositions are packaged in containers wherein the mixture is dispensed through an atomizing valve.
  • Propellant mixtures contain low boiling halogenated hydrocarbons (which may be mixed with organic solvents) or aqueous suspensions under pressure with inert gases or gaseous hydrocarbons.
  • the present disclosure provides a method for preparing the aforementioned insecticidal composition, the preparation method comprising inoculating the strain into a medium for fermentation and culturing to prepare the insecticidal composition.
  • the medium includes seed medium and fermentation medium.
  • the composition of the seed medium is: 0.8-1.2 parts by mass of glucose, 0.8-1.2 parts by mass of yeast extract, 0.16-0.24 parts by mass of N-Z amine type A, 1.8-2.4 parts by mass of corn starch, 0.08-0.12 parts by mass of ammonium sulfate, 2-3 parts by mass of cottonseed meal, 0.16-0.24 parts by mass of magnesium sulfate heptahydrate, water of 80-120 parts by mass; preferably, the water is distilled water more preferably, the composition of the seed culture medium is: the glucose of 1 mass part, the yeast extract of 1 mass part, the N-Z amine typeA of 0.2 mass part, the cornstarch of 2 mass parts, the ammonium sulfate of 0.1 mass part, 2.5 parts by mass of cottonseed cake powder, 0.2 parts by mass of magnesium sulfate heptahydrate, and 100 parts by mass of water; preferably, the pH value of the medium is 6.8-7.2, preferably 7.0.
  • the composition of the fermentation medium is: 6.4-9.6 parts by mass of glucose, 0.4-0.6 parts by mass of yeast powder, 0.8-1.2 parts by mass of protein powder, 1.6-2.4 parts by mass of cottonseed cake powder, 0.32-0.48 parts by mass of trisodium citrate, 0.16-0.24 parts by mass of dipotassium hydrogen phosphate, 0.24-0.36 parts by mass of calcium carbonate, 0.16-0.24 parts by mass of ammonium sulfate, 4-6 parts by mass of rapeseed oil, 80-120 parts by mass of water; preferably, the water is distilled water; more preferably, the composition of the fermentation medium is: 8 parts by mass of glucose, 0.5 parts by mass of yeast powder, 1 part by mass of protein powder, 2 mass parts of cottonseed cake flour, 0.4 mass parts of trisodium citrate, 0.2 mass parts of dipotassium hydrogen phosphate, 0.3 mass parts of calcium carbonate, 0.2 mass parts of ammonium sulfate, 5 mass
  • the fermentation conditions are 200-300rpm, 10-40°C, and 50-70% humidity.
  • the fermentation conditions are 250rpm, 28°C, and 60% humidity.
  • the present disclosure provides the use of the aforementioned insecticidal composition in the preparation of drugs for killing insects, killing acaria, and killing nematodes.
  • the insecticidal, acaricidal, and nematicidal drugs can be applied to animals related to agriculture, such as cattle, pigs, sheep, goats, horses or donkeys, or plants , such as soybeans, corn, sugar cane, canola, potatoes, cotton, rice, coffee, citrus, almonds, tea.
  • the medicament may be enterally administered to animals, such as in the form of tablets, capsules, drinks, drenches, granules, pastes, boluses, and suppositories.
  • the drug is administered to the animal parenterally, such as by injection (including intramuscular, subcutaneous, intravenous, intraperitoneal, etc.), implantation, showering or dipping.
  • the drug is administered to plants by spraying, pouring, dripping, washing, or dusting.
  • the compounds of the present invention can be used for pest control on various plants including soybean (for example 10-70 g/ha in some cases), corn (for example 10-70 g/ha in some cases), sugarcane (for example 20-200g/ha in some cases), alfalfa (eg 10-70g/ha in some cases), brassica (eg 10-50g/ha in some 20-70g/ha in some cases), potato (including sweet potato) (for example 10-70g/ha in some cases), cotton (for example 10-70g/ha in some cases), rice (for example in some cases 10-70g/ha), coffee (eg 30-150g/ha in some cases), citrus (eg 60-200g/ha in some cases), almonds (eg 40-180g/ha in some cases ha), fruit vegetables (e.g.
  • soybean for example 10-70 g/ha in some cases
  • corn for example 10-70 g/ha in some cases
  • sugarcane for example 20-200g/ha in some cases
  • alfalfa eg 10-70g/ha in some
  • tomato, pepper, capsicum, eggplant, cucumber, pumpkin, etc. (e.g. in some cases 10-80g/ha), tea (e.g. in some cases 20-150g/ha ), bulb vegetables (e.g. onions, leeks, etc.) (e.g. 30-90g/ha in some cases), grapes (e.g. 30-180g/ha in some cases), pome fruits (e.g. apples, pears, etc.) (eg 30-180g/ha in some cases) and stone fruit (eg pears, plums etc.) (eg 30-180g/ha in some cases).
  • bulb vegetables e.g. onions, leeks, etc.
  • grapes e.g. 30-180g/ha in some cases
  • pome fruits e.g. apples, pears, etc.
  • stone fruit eg pears, plums etc.
  • pesticidal compositions are useful in the field of animal health, eg they are useful against parasitic invertebrate pests, more preferably against parasitic invertebrate pests in or on animals.
  • the animal may be a non-human animal, such as an agriculturally related animal such as a cow, pig, sheep, goat, horse or donkey.
  • the dilution method is used to separate S. spinosa in soil.
  • the basic formula of the separation medium is: add 3g of yeast extract, 10g of enzymatic casein (N-Z amine type A), and 5g of glucose to 1L of distilled water, mix and dissolve, adjust the pH to 7.0 with NaOH; Pack each bottle, add 4g of agar to each bottle; sterilize at 115°C for 30min. After the sterilization was completed, the medium was cooled to about 65°C, and nystatin was added to a final concentration of 50 ⁇ g/mL and nalidixic acid to a final concentration of 25 ⁇ g/mL to inhibit the growth of fungi and Gram-negative bacteria, respectively.
  • each bottle of bacteria was made in three parallels. Secondary seeds were transferred and fermented according to the transferred amount of 5%, and each bottle of seeds was set up in three parallels; the fermentation culture conditions were: cultivated for 12 days at 250rpm, 28°C, and 60% humidity.
  • the culture medium used for the primary seed and the secondary seed is a seed medium.
  • Seed medium add 1g of glucose, 1g of yeast extract, 0.2g of N-Z amine type A, 2g of cornstarch, 0.1g of ammonium sulfate, 2.5g of cottonseed cake powder, 0.2g of Magnesium sulfate heptahydrate, mix well and dissolve, then adjust the pH to 7.0 with NaOH; for seed culture, fill each bottle with 25mL seed medium; sterilize at 121°C for 30min.
  • Fermentation medium add 8g of glucose, 0.5g of yeast powder, 1g of protein powder, 2g of cottonseed cake powder, 0.2g of ammonium sulfate, 0.2g of dipotassium hydrogen phosphate, 0.4g of Trisodium citrate, 0.3g of calcium carbonate, 5g of rapeseed oil; mix and dissolve and adjust the pH to 7.0 with NaOH; Fill each bottle.
  • the first round of mutagenesis was carried out by irradiating at a distance of 20 cm from a 15W ultraviolet lamp for 30 s, and then gradually diluting and coating the plate according to a 10-fold decrease.
  • the second round of mutagenesis adopts the method of chemical mutagenesis, that is, adjust the concentration of spores to 10 5 -10 6 /mL, take 1 mL into a new centrifuge tube , add N-methyl-N'nitro-N-nitrosoguanidine (MNNG) solution to make the mutagenic concentration 1mg/mL MNNG, after co-incubating for half an hour, wash the spores repeatedly three times with sterile water and then carry out gradient Dilute the plate.
  • MNNG N-methyl-N'nitro-N-nitrosoguanidine
  • strains to be screened After repeated 6 rounds of mutagenesis (3 rounds of ultraviolet mutagenesis, 3 rounds of chemical mutagenesis, ultraviolet mutagenesis and chemical mutagenesis were carried out alternately), the highest group of 20 strains was selected as strains to be screened and named W600.
  • the 20 strains to be screened named W600 obtained in the last round were fermented one by one.
  • spinosyn A and spinosyn D could be detected in the fermentation broth (Fig. 1).
  • the 16S rRNA gene sequence of bacterial strain W618 was amplified by PCR, sequenced, and the nucleotide sequence of its 16S rRNA gene was obtained, and the specific sequence was shown in SEQ ID NO:1. According to Blast comparison, the 16S rRNA gene of W618 has the highest sequence similarity with GENBANK ACCESSION CP061007.1, with a similarity of 99.93%. According to the above results, the strain W618 was identified as belonging to Saccharopolyspora spinosa.
  • Saccharopolyspora spinosa is an aerobic Gram-positive actinomycete that can grow well on most media (such as YMS, calcium malate, etc.), forming aerial mycelia and white spores.
  • the suitable growth temperature for bacteria is 25°C to 35°C.
  • strain W618 The morphology of strain W618 is shown in Figure 4.
  • the bacterial strain of the present invention is Saccharopolyspora spinosa.
  • the strain W618 was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on October 12, 2021.
  • CCTCC NO: M 20211261 The preservation number is CCTCC NO: M 20211261, and the classification is named Saccharopolyspora spinosa. (Saccharopolyspora spinosa).
  • the wild-type strain of S. spinosa NRRL18395 (American Agricultural Research Culture Collection Center) was obtained, and the wild-type S. spinosa was fermented and cultured according to the fermentation method in Example 1.
  • the yield of spinosyn produced by fermentation of S. spinosa W618 provided by the present disclosure is as high as about 6 g/L.
  • the yield of the wild-type S. spinosa was 83.5 mg/L.

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Abstract

提供了一种高产多杀菌素菌株及其用途，该菌株的保藏号为CCTCC NO:M20211261，该菌株可以生产多种多杀菌素，且产量高。

Description

高产多杀菌素菌株及其用途 技术领域
本公开属于生物发酵技术领域，具体而言，本公开涉及一种高产多杀菌素菌株及其用途。
背景技术
农药作为农业生产中必不可少的生产资料，对农业发展和人类粮食供给做出了巨大的贡献。农药主要包括杀菌剂、杀虫剂和除草剂三大类。世界上有成千上万种由化学药品作为活性成分的制剂化的杀虫剂产品。上述杀虫剂产品包括抗菌剂、杀螨剂、杀菌剂、杀线虫剂、动物药浴剂、杀害鸟剂和消毒剂。化学防治具有见效快效果显著使用方便、不受地区和季节限制等优势适，用于大面积防治目前仍然是害虫防治的主要手段。
然而化学杀虫剂的长期大量使用在控制有害生物维护人类利益的同时也导致了一系列严重问题，包括广泛的昆虫抗性、次要害虫的出现、对人和动物健康的危害以及对鱼和鸟类的有害影响、环境污染和新杀虫剂经济成本的增长。
对于人体来说，农药往往通过皮肤、呼吸系统和消化系统造成伤害，可以带来对杀虫剂的急性毒性反应如中毒、皮肤和眼睛刺激等，以及可能的长期影响，如引起生物体内细胞产生基因突变，改变遗传细胞机制，导致致癌或发生畸形等。
根据中国农药毒性分级标准，多杀菌素属低毒杀虫剂。多杀菌素属广谱杀虫剂，能有效地控制鳞翅目、双翅目和缨翅目的害虫，可以很好地防治鞘翅目和直翅目中某些大量吞食叶片的害虫种类。它对哺乳动物和鸟类相对低毒，对水生动物也只是轻微的中等毒性。综合上述因素，在进行害虫综合治理时，多杀菌素通常是首选。
目前，多杀菌素仍由刺糖多孢菌通过有氧发酵进行生产。尽管有多个专利报道通过改良培养基或发酵罐的控制方法来提升多杀菌素的产量，但由于菌种本身发酵产量不佳，因此通过发酵优化也很难实现规模化生产。
因此，急需寻找新的高产多杀菌素的菌株以解决多杀菌素产量低等问题。
发明内容
为了克服现有技术上的缺陷，本公开的目的在于提供一种新的高产多杀菌素菌株，其可以生产多种多杀菌素，且产量高，其中，多杀菌素A和多杀菌素D的摇瓶产量总和约为6g/L。
在一方面，本公开提供一种高产多杀菌素菌株，其为糖多孢菌属的土壤放线菌，即刺糖多孢菌(Saccharopolyspora spinosa)W618，其保藏号为CCTCC NO:M 20211261。
在另一方面，本公开提供一种杀虫组合物，所述组合物包含所述菌株。
在一些实施方案中，所述组合物还包含选自以下一种或多种化合物：有机磷化合物、氨基甲酸酯类化合物、合成除虫菊酯类化合物、酰基脲化合物、其他类型的昆虫生长调节剂和昆虫激素类似物、类新烟碱和其他烟碱类化合物、大环内酯化合物、其他杀昆虫、杀螨、杀软体动物与杀线虫的化合物或活性成分和吞噬刺激剂。
在一些实施方案中，所述杀虫组合物可还包含药学上可接受的载体，如液体载体、或固体载体，
在一些实施方案中，所述液体载体可选自脂肪酮类(如环己酮)和复合醇类(如2-乙氧基乙醇)和芳香化合物，特别是二甲苯和石油馏分，特别是石油的高沸点的萘类和烯类部分如重芳香石油脑中的一种或多种。
在一些实施方案中，所述固体载体可选自硅镁土黏土、蒙脱石黏土、硅藻土或纯化的硅酸盐中的一种或多种。
在一些实施方案中，所述杀虫组合物的剂型可选自可湿性粉剂、可溶性粉剂、乳油、水悬浮剂、可分散油悬浮剂、水乳剂、气雾剂、微乳剂和水分散颗粒剂。
在另一方面，本公开提供了前述杀虫组合物的制备方法，所述制备方法包括将前述菌株接种到培养基中进行发酵培养来制备所述杀虫组合物。
在一些实施方案中，所述培养基包括种子培养基和发酵培养基。
在一些实施方案中，所述种子培养基和/或发酵培养基用pH调节剂调节pH；优选地，pH调节剂是碱；更优选地，所述碱是氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠中的一种或多种。
在一些实施方案中，所述发酵的条件为200-300rpm、10-40℃、湿度为50-70％；
在一些实施方案中，所述发酵条件为250rpm、28℃、湿度60％。
在另一方面，本公开提供了前述杀虫组合物在制备杀昆虫、杀蜱螨亚纲动物、杀线虫的药物的用途。
在一些实施方案中，所述杀昆虫、杀蜱螨亚纲动物、杀线虫的药物可以施用于与农业有关的动物，例如牛、猪、绵羊、山羊、马或驴，或植物，例如大豆、玉米、甘蔗、油菜、马铃薯、棉花、水稻、咖啡、柑橘、扁桃、茶。
在一些实施方案中，所述药物可以为对动物通过肠内给药的药物，例如片剂、胶囊、饮品、灌服、颗粒剂、糊剂、大丸剂、栓剂形式的药物。
在一些实施方案中，对动物通过肠胃外给药的药物，例如通过注射(包括肌内、皮下、静脉内、腹膜内等等)、植入、淋浴、浸渍、喷雾、倾倒、沾滴、洗涤、撒粉的形式来进行应用的药物。
在一些实施方案中，对植物通过喷雾、倾倒、沾滴、洗涤、撒粉的形式来给药的药物。
刺糖多孢菌(Saccharopolyspora spinosa)W618，已于2021年10月27日保藏于中国典型培养物保藏中心(简称CCTCC，地址为中国，武汉，武汉大学)，保藏号为CCTCC NO:M 20211261。
含有刺糖多孢菌W618和/或其发酵产物和/或其菌悬液和/或其培养液的杀虫组合物，能有效地控制鳞翅目、双翅目和缨翅目的害虫，可以很好地防治鞘翅目和直翅目中某些大量吞食叶片的害虫种类。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
图1显示了利用LC-MS检测发酵物的成分的结果，其中，上图为检测实施例1中发酵物的结果，下图为标准品多杀菌素的检测结果。
图2显示了利用LC检测发酵物中各种多杀菌素的结果，发酵物中除了能检测到多杀菌素A和多杀菌素D，还能检测到多杀菌素A-iso,多杀菌素B、多杀菌素E、多杀菌素H、多杀菌素J、多杀菌素K、多杀菌素L、多杀菌素L2、多杀菌素L3、多杀菌素P、多杀菌素P-CH 2。
图3显示了6轮诱变后筛选刺糖多孢菌菌株W600中20株菌株进行逐一发酵后的产量结果，其中最高产量的刺糖多孢菌菌株为W618。
图4示出了刺糖多孢菌菌株W600种子培养成熟的菌落照片。
具体实施方式
定义
本公开中所述的术语“发酵产物”是指通过包括使用发酵生物进行的发酵步骤在内的方法生产的产物。
本公开中所述的术语“菌悬液”是指在发酵生物发酵结束后，发酵液经生物分离提取技术进行固液分离所收集获得的混合物，常用离心或膜过滤进行分离收集。
本公开中所述的术语“培养液”是指含有发酵生物和培养基二者的液体培养物。
本公开中所述术语的“植物”包括苗、灌木和树木。植物应被理解为还包括已经通过常规的育 种或通过基因工程的方法被赋予对除草剂或多种类别的除草剂(例如ALS-、GS-、EPSPS-、PP0-以及HPPD-抑制剂)耐受性的那些作物。植物还应理解为是通过常规的育种或基因工程的方法获得并且包括所谓的输出型(output)性状(例如改进的储存能力、更高的营养价值以及改进的香味)。
本公开中所述术语的“杀虫组合物”是指针对害虫具有杀虫作用的组合物。
如关于兽医领域所使用的，术语“控制”指杀虫组合物将一种受这类寄生虫感染的动物的相应寄生虫的发生率有效地降低到无害水平。更具体地，如在此使用的，“控制”指这些活性化合物在杀死相应寄生虫、抑制它的生长或抑制它的繁殖方面是有效的。总体而言，当用于动物处理时，根据本发明的这些杀虫组合物可以直接施用。优选地，这些杀虫组合物可以包含药学上可接受的载体和/或本领域内知晓的助剂。
可以被本公开所述的“杀虫组合物”控制的具体代表性节肢动物害虫包括如下：美洲花蜱(Amblyomma americanum)，有斑花蜱(Amblyomma maculatum)波斯锐缘蜱(Argas persicus)，微小牛蜱(Boophilus microplus)，安氏革蜱(Dermacentor anderson)，变形革蜱(Dermacentor variabilis)，鸡皮刺螨(Dermanyssus gallinae)，蓖子硬蜱(Ixodes ricinus)，血红扇头蜱(Rhipicephalus sanguineus)，痒螨属(Chorioptes spp.)，牛螨形螨(Demodex bovis)，犬蠕形螨(Demodex canis)，鸡疙螨(Knemidokoptes gallinae)，禽疙螨(Knemidokoptes mutans)，Otobius megnini(ear tick)，马瘙螨(Psoroptes equi)，绵羊瘙螨(Psoroptes ovis)，人疥螨(Sarcoptes scabiei)，伊蚊属(Aedes)，按蚊属(Anopheles)，库蚊属(Culex)，脉毛蚊属(Culiseta)，Bovicola bovis(cattle biting louse)，Callitroga homnivorax(blowfly)，斑虻属(Chrysops spp).(deer fly)，温带臭虫(Cimex lectularius)，犬栉首蚤(Ctenocephalides canis)，猫蚤(Ctenocephalides felis)，库蠓属(Culicoides spp).(midges，sandflies，punkies，or no-see-ums)，绵羊畜虱(Damaliniaovis)，锥蝇属(Cochliomyiaspp)，肤蝇属(Dermatobia spp)，赤尾胃蝇(Gasterophilushaemorrhoidalis)，肠胃蝇(Gasterophilus intestinalis)，鼻胃蝇(Gasterophilus nasalis)，舌蝇属(Glossina spp.)，西方角蝇(Haematobia irritans)，牛皮蝇(Hypoderma bovis)，纹皮蝇(Hypoderma lineatum)，绿蝇属(Lucilia spp.)，羊蜱蝇(Melophagus ovinus)，家蝇(Musca spp.)，羊狂蝇(Oestrus ovis)，伏蝇(Phormia regina)，厩螯蝇(Stomoxys calcitrans)，Hydrotaeairritans(head fly)，Linognathus ovillus(body louse)，Linognathus pedalis(foot louse)，牛颚虱(Linognathusvituli)，驴血虱(Haematopinus asini)，牛血虱(Haematopinus eurysternus)，Haematopinus ovillus(bodylouse)，猪血虱(Haematopinus suis)，虱(Pediculus spp.)，牛管虱(Solenopotes capillatus)，白蛉(Phlebotomus spp.)，Psorophora spp.(mosquito)，Pthirus spp.(lice)，猪蝽(Reduvius spp.)，蚋(Simulium spp.)，虻(Tabanus spp.)，粉甲属(Tenebriospp.)，椎猎蝽(Triatoma spp.)。本公开所提供的杀虫组合物可用于防治体内寄生虫和体表寄生虫。这些杀虫组合物以已知的方式(通过以例如片剂、胶囊、饮品、灌服、颗粒剂、糊剂、大丸剂、栓剂形式肠内给药；通过给动物肠胃外给药或植物给药，例如像通过注射(包括肌内、皮下、静脉内、腹膜内等等)、植入，通过以例如淋浴或浸渍、喷雾、倾倒和沾滴、洗涤、撒粉的形式以及在包含杀虫组合物的成型物品(例如，辊环、耳标、尾标、肢体带、缰绳、制造装置以及类似物)的帮助下的皮肤施用)来进行应用(例如给药)。
现有的制备多杀菌素类化合物的方法，利用刺糖多孢菌生产多杀菌素类化合物的产量较低，导致工业化制备多杀菌素类化合物的成本较高。发明人从大量的菌株中通过产多杀菌素的条件筛选以及紫外多轮诱变，获得一种多杀菌素高产菌株刺糖多孢菌(Saccharopolyspora spinosa)W618，利用其发酵制备多杀菌素摇瓶产量高达6g/L。因此，刺糖多孢菌W618可作为多杀菌素高产的工业化菌株进行开发。
在一方面，本公开提供一种高产多杀菌素菌株，其为糖多孢菌属的土壤放线菌，即刺糖多孢菌(Saccharopolyspora spinosa)W618，其保藏号为CCTCC NO:M 20211261。
在另一方面，本公开提供了一种杀虫组合物，所述组合物包含前述刺糖多孢菌。
在一些实施方案中，所述杀虫组合物包含多杀菌素A、多杀菌素A-iso、多杀菌素B、多杀菌素C、多杀菌素D、多杀菌素E、多杀菌素F、多杀菌素G、多杀菌素H、多杀菌素J、多杀菌素 K、多杀菌素L、多杀菌素L2、多杀菌素L3、多杀菌素P、多杀菌素P-CH 2等30余种结构类似组分中的一种或多种。
在一些优选的实施方案中，所述杀虫组合物包含多杀菌素A和多杀菌素D。
在一些实施方案中，所示杀虫组合物还包含选自以下一种或多种化合物：有机磷化合物、氨基甲酸酯类化合物、合成除虫菊酯类化合物、酰基脲化合物、其他类型的昆虫生长调节剂和昆虫激素类似物、类新烟碱和其他烟碱类化合物、大环内酯化合物、其他杀昆虫、杀螨、杀软体动物与杀线虫的化合物或活性成分和吞噬刺激剂。
在一些优选的实施方案中，所述有机磷化合物选自以下一种或多种化合物：高灭磷、谷硫磷、硫线磷、壤虫氯磷、毒死蜱、蝇毒磷、dematon、内吸磷、二嗪农、敌敌畏、乐果、EPN、erthoate、灭克磷、氧嘧啶磷、克线磷、杀螟松、丰索磷、倍硫磷、地虫磷、安果、噻唑酮磷、蚜螨磷(庚虫磷)、马来硫磷、甲胺磷、甲基对硫磷(甲基一六0五)、速灭磷、久效磷、对硫磷(一六0五)、甲拌磷、伏杀磷、亚胺硫磷、磷胺、乙丙磷威、肟硫磷(腈肟磷)、丙溴磷、丙虫磷、烯虫磷、丙硫磷、甲基虫螨磷、乙基虫螨磷、喹硫磷(喹噁磷)、硫灭克磷(乙丙硫磷)、嘧丙磷、双硫磷、特丁磷、杀虫畏、thiafenox、二甲硫吸磷(甲基乙拌磷)、三唑磷和敌百虫。
在一些优选的实施方案中，所述氨基甲酸酯类化合物选自以下一种或多种化合物：涕灭威、噁虫威、丙硫克百威、杀虫磺、BPMC、氧丁叉威(butoxycarbocim)、甲萘威(西维因)、呋喃丹(虫螨威)、丁硫克百威、除线威、苯虫威、丁苯威、呋线威、灭虫威、异丙威、灭多虫、甲氨叉威、抗蚜威、猛杀威、残杀威、硫双灭多威和thiofurox。
在一些优选的实施方案中，所述合成除虫菊酯类化合物选自以下一种或多种化合物：氟酯菊酯、丙烯菊酯、β-氟氯氰菊酯、氟氯菊酯、右旋反灭虫菊酯、氟氯氰菊酯、格林奈、λ-格林奈、γ-格林奈、氯氰菊酯、α-氯氰菊酯、ζ-氯氰菊酯、溴氰菊酯、高氰戊菊酯、杀灭菊酯、五氟苯菊酯、甲氰菊酯、氟氰戊菊酯、氟氯苯菊酯、氯氟胺氰戊菊酯、τ-氯氟胺氰戊菊酯、卤醚菊酯、氯菊酯、protrifenbute、灭虫菊、灭虫硅醚、七氟菊酯、胺菊酯、四溴菊酯、对鱼类安全的类除虫菊酯，例如醚菊酯、天然除虫菊酯类、胺菊酯、S-反丙烯除虫菊、五氟苯菊酯和炔酮菊酯。
在一些优选的实施方案中，所述酰基脲、其他类型的昆虫生长调节剂和昆虫激素类似物选自以下一种或多种化合物：噻嗪酮、chromfenozide、定虫隆、伏虫脲(氟脲杀)、双氧威、氟虫脲、特丁苯酰肼、氟铃脲、蒙五一二、氟丙氧脲、蒙五一五、甲氧苯酰肼、双苯氟脲、蚊蝇醚、伏虫隆和双苯酰肼、N-[3，5-二氯-2-氟-4-(1，1，2，3，3，3-六氟丙氧基)苯基]-N’-(2，6-二氟苯甲酰基)脲。
在一些优选的实施方案中，所述类新烟碱和其他烟碱类选自以下一种或多种化合物：吡虫清、AKD-1022、巴丹、TI-435、clothiamidin、MTI-446、dinotefuran、吡虫啉、烟碱、硝胺烯啶、thiamethoxam、thiacloprid。
在一些优选的实施方案中，所述大环内酯类化合物选自以下一种或多种化合物阿凡曼菌素、米尔倍霉素、阿巴美丁、伊维菌素、米尔倍霉素和emamectin benzoate。
在一些优选的实施方案中，所述其他杀昆虫、杀螨、杀软体动物与杀线虫的化合物或活性成分选自以下一种或多种化合物：阿耳德林、虫螨脒、艾扎丁、唑环锡、联苯肼酯、溴螨酯、氯苯甲脒、氯唑虫清、chlofentezine、乙酯杀螨醇、氯丹、三环锡、灭蝇胺、DDT、三氯杀螨醇、狄氏剂、DNOC、硫丹、ethoxazole、喹螨醚、杀螨锡、唑螨酯、β-唑螨酯、锐劲特、氟苯亚胺噻唑、噻螨酮、IKI-220、噁二唑虫、林丹、灭虫威、蜗牛敌、甲氧滴滴涕、neem、石油与植物油、哒螨酮、拒嗪酮、嘧胺苯醚、鱼藤酮、S-1812、S-9539、spirodiclofen、硫磺、吡螨胺、三氯杀螨砜、唑蚜威、昆虫活性的植物提取物、含有昆虫活性线虫的制备物、可从枯草芽孢杆菌、苏云金芽孢杆菌、核多角体病毒或其他经过遗传修饰或天然生物得到的制备物，以及增效剂，例如胡椒基丁醚、sesamax、safroxan和十二烷基咪唑。
在一些优选的实施方案中，所述吞噬刺激剂选自以下一种或多种化合物：葫芦素、糖和Coax。
在一些实施方案中，所述杀虫组合物可以还包含药学上可接受的载体，如液体载体、或固体载体。
所述液体载体可选自脂肪酮类(如环己酮)和复合醇类(如2-乙氧基乙醇)和芳香化合物，特别是二甲苯和石油馏分，特别是石油的高沸点的萘类和烯类部分如重芳香石油脑中的一种或多种。
所述固体载体可选自硅镁土黏土、蒙脱石黏土、硅藻土或纯化的硅酸盐中的一种或多种。
在一些优选的实施方案中，所述杀虫组合物的剂型选自可湿性粉剂、可溶性粉剂、乳油、水悬浮剂、可分散油悬浮剂、水乳剂、气雾剂、微乳剂和水分散颗粒剂。
本文中描述的杀虫组合物可与药学上可接受的载体一起提供以提供，例如饵剂、浓缩乳剂、粉剂、乳化浓缩剂、熏剂、凝胶剂、颗粒剂、微囊剂、种子处理剂、悬浮液浓缩剂、悬浮乳剂、片剂、水溶性液体、水分散颗粒剂或干燥可流动的可湿性粉剂和超低体积溶液剂。
杀虫组合物的含水混悬剂或乳剂可以频繁地使用。这样的水溶性、水悬浮性或可乳化的制剂是固体(通常被认为是可湿性粉剂或水分散颗粒剂)或液体(通常被认为是乳化浓缩剂、或水性悬浮剂)。可被压实以形成水分散颗粒剂的可湿性粉剂包含杀虫组合物、载体和表面活性剂的亲密混合物。所述载体通常选自硅镁土黏土、蒙脱石黏土、硅藻土或纯化的硅酸盐。有效的表面活性剂(占可湿性粉剂的大约0.5％至大约10％)选自磺化木质素、稠合萘磺酸盐、萘磺酸盐、烷基苯磺酸盐、烷基磺酸盐和非离子表面活性剂如烷基苯酚的环氧乙烷加合物。
所要求保护的杀虫组合物的可乳化浓缩物通常为约50至约500克组合物每升液体，等于约10％至约50％，溶解在惰性载体中，后者是水不可混溶性溶剂与乳化剂的混合物。有机溶剂包括有机物，例如二甲苯，和石油级分，例如石油的高沸点萘属与烯属部分，包括重石脑油和芳族石脑油。还可以使用其他有机物，例如萜烯溶剂-松香衍生物、脂族酮，例如环己酮，和复合醇。用于可乳化浓缩物的乳化剂通常是混合的离子和/或非离子表面活性剂，例如本文提到的那些或它们的等价物。
水悬液可以制成含有本发明的杀虫组合物的水不溶性化合物，其中这些化合物分散在水性赋形剂中的浓度通常在约5至约50重量％的范围内。悬液是通过微细研磨杀虫组合物，剧烈混合在水、表面活性剂与分散剂的赋形剂中而制备。根据需要，还可以采用惰性成分，例如无机盐和合成或天然树胶，以增加水性赋形剂的密度和/或粘度。
沉淀性流体可以这样制备，将活性分子溶于水可混溶性溶剂和表面活性剂或表面活性聚合物中。在将这些制剂与水混合时，活性化合物沉淀出来，而表面活性剂控制所得微晶沉淀的大小。晶体的大小可以通过选择特定的聚合物与表面活性剂混合物加以控制。
杀虫组合物还可以制备成颗粒剂施用于土壤。杀虫组合物是分散在惰性载体中的，后者通常是粘土或等价物。一般而言，颗粒剂是这样制备的，将杀虫组合物溶于适合的溶剂，涂在颗粒状载体上，后者已被加工成所需的粒径。粒径通常在约0.5mm至3mm之间。颗粒剂还可以这样制备，使载体与杀虫组合物形成面团或糊，干燥混合物，再将面团或糊粉碎成所需的粒径。
这些杀虫组合物还可以与适当的有机溶剂混合。有机溶剂通常是广泛用在农业中的温和溶剂。这些组合通常被用作喷雾剂。更普遍地，将杀虫组合物作为在液体载体中的分散系而施用，其中该液体载体是水。杀虫组合物还可以以气雾剂组合物的方式施用。所述杀虫组合物是溶解在惰性载体中的，该载体是产生压力的推进剂混合物。气雾剂组合物是包装在容器内的，其中通过雾化阀分配混合物。推进剂混合物含有低沸点卤代烃(它们可以与有机溶剂相混合)或用惰性气体或气态烃施加压力的水悬液。
在另一方面，本公开提供了前述杀虫组合物的制备方法，所述制备方法包括所述的菌株接种到培养基中进行发酵培养来制备所述杀虫组合物。
在一些实施方案中，所述培养基包括种子培养基和发酵培养基。
在一些优选的实施方案中，所述种子培养基的组成为：0.8-1.2质量份的葡萄糖、0.8-1.2质量份的酵母提取物、0.16-0.24质量份的N-Z amine typeA、1.8-2.4质量份的玉米淀粉、0.08-0.12质量份的硫酸铵、2-3质量份的棉籽饼粉、0.16-0.24质量份的七水硫酸镁、80-120质量份的水；优选地，所述水为蒸馏水；更优选地，所述种子培养基的组成为：1质量份的葡萄糖、1质量份的酵母提取物、0.2质量份的N-Z amine typeA、2质量份的玉米淀粉、0.1质量份的硫酸铵、2.5质量份的棉籽饼粉、0.2质量份的七水硫酸镁，100质量份的水；优选地，所述培养基的pH值为 6.8-7.2，优选为7.0。
在一些优选的实施方案中，所述发酵培养基的组成为：6.4-9.6质量份的葡萄糖、0.4-0.6质量份酵母粉、0.8-1.2质量份的蛋白粉、1.6-2.4质量份的棉籽饼粉、0.32-0.48质量份的柠檬酸三钠、0.16-0.24质量份的磷酸氢二钾、0.24-0.36质量份的碳酸钙、0.16-0.24质量份的硫酸铵、4-6质量份的菜籽油、80-120质量份的水；优选地，所述水为蒸馏水；更优选地，所述发酵培养基的组成为：8质量份的葡萄糖、0.5质量份的酵母粉、1质量份的蛋白粉、2质量份的棉籽饼粉、0.4质量份的柠檬酸三钠、0.2质量份的磷酸氢二钾、0.3质量份的碳酸钙、0.2质量份的硫酸铵、5质量份的菜籽油、100质量份的水；优选地，所述培养基的pH值为6.8-7.2，优选为7.0。
在一些实施方案中，根据前述制备方法，其中，所述发酵的条件为200-300rpm、10-40℃、湿度为50-70％。
在一些优选的实施方案中，所述发酵条件为250rpm、28℃、湿度60％。
在另一方面，本公开提供了前述杀虫组合物在制备杀昆虫、杀蜱螨亚纲动物、杀线虫的药物的用途。
在一些实施方案中，根据前述用途，所述杀昆虫、杀蜱螨亚纲动物、杀线虫的药物可以施用于与农业有关的动物，例如牛、猪、绵羊、山羊、马或驴，或植物，例如大豆、玉米、甘蔗、油菜、马铃薯、棉花、水稻、咖啡、柑橘、扁桃、茶。
在一些实施方案中，所述药物可以为对动物通过肠内给药的药物，例如片剂、胶囊、饮品、灌服、颗粒剂、糊剂、大丸剂、栓剂形式的药物。
在一些实施方案中，对动物通过肠胃外给药的药物，例如通过注射(包括肌内、皮下、静脉内、腹膜内等等)、植入、淋浴或浸渍的形式来进行应用的药物。
在一些实施方案中，对植物通过喷雾、倾倒、沾滴、洗涤、撒粉的形式来给药的药物。
本发明的这些化合物可以用于对不同植物进行有害生物控制，包括大豆(例如在某些情况下10-70g/ha)、玉米(例如在某些情况下10-70g/ha)、甘蔗(例如在某些情况20-200g/ha)、苜蓿(例如在某些情况下10-70g/ha)、芸薹(例如在某些情况下10-50g/ha)、油菜(例如卡罗拉(例如在某些情况下20-70g/ha)、马铃薯(包括甘薯)(例如在某些情况10-70g/ha)、棉花(例如在某些情况下10-70g/ha)、水稻(例如在某些情况下10-70g/ha)、咖啡(例如在某些情况下30-150g/ha)、柑橘(例如在某些情况下60-200g/ha)、扁桃(例如在某些情况下40-180g/ha)、果实类蔬菜(例如番茄、胡椒、番椒、茄子、黄瓜、南瓜、等)(例如在某些情况下10-80g/ha)、茶(例如在某些情况下20-150g/ha)、鳞茎类蔬菜(例如洋葱、韭等)(例如在某些情况下30-90g/ha)、葡萄(例如在某些情况下30-180g/ha)、梨果(例如苹果、梨等)(例如在某些情况下30-180g/ha)以及核果(例如梨、李子等)(例如在某些情况下30-180g/ha)。
这些杀虫组合物可用于动物健康领域，例如它们可用于对抗寄生无脊椎动物有害生物，更优选用于对抗在动物体内或动物体表的寄生无脊椎动物有害生物。该动物可为非人类动物，例如与农业有关的动物，例如牛、猪、绵羊、山羊、马或驴。
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
实施例1：高产多杀菌素的刺糖多孢菌的获取
1、产多杀菌素菌株的获取
(1)菌株分离
本实施例采用稀释法来分离土壤中的刺糖多孢菌。
称取从山东省不同地区采集的土壤10g，放入装有无菌水的250ml三角瓶中，充分震荡10min，制成0.1g/mL土壤悬浮液，并将10颗玻璃珠加入到土壤悬浮液中。然后将其按照递减10倍进行逐级稀释，用每个浓度的溶液涂布10块分离培养基。在最终长出来的菌落中，挑选菌落形态为产生孢子的链霉菌样样本进行保种，共收集382株菌，获得分离的菌株。
其中，分离培养基的基础配方为：在1L的蒸馏水中加入3g酵母提取物、10g酶解酪蛋白(N-Z amine type A)、5g葡萄糖，混合充分溶解后用NaOH调节至pH为7.0；按照200mL/瓶进行分装，每瓶加入4g琼脂；115℃灭菌30min。灭菌完成后将培养基冷却至65℃左右，加入制霉菌素至终浓度50μg/mL和萘啶酮酸至终浓度25μg/mL，以分别抑制真菌和革兰氏阴性菌生长。
(2)产多杀菌素菌株的获取
将上述382株菌中每10株菌进行混合发酵，发酵方法如下：
(A)发酵培养
将每个分离菌株挑取单菌落划菌于另一新的分离培养基平板生长，待生长到适合状态，划取1cm×1cm左右大小的菌块于种子培养基中，10株分离菌株接种于一个培养瓶，250rpm、28℃、60％的湿度下培养一级种子96h后镜检观察一级种子是否染菌，以及种子的生长状态，未染菌且生长状态良好则按1％的转接量转接二级种子；二级种子培养条件与一级种子相同，培养到60h的时候进行二级种子镜检并观察种子是否染菌，若正常则进行发酵的转接，按5％的转接量转接，每瓶菌做三个平行。二级种子按5％的转接量转接发酵，每瓶种子设三个平行；发酵培养条件为：在250rpm、28℃、湿度60％的条件下培养12天。其中，一级种子和二级种子所使用的培养基均为种子培养基。
其中，种子培养基和发酵培养基的配方如下：
(a)种子培养基：每100mL蒸馏水中加入1g的葡萄糖、1g的酵母提取物、0.2g的N-Z amine typeA、2g的玉米淀粉、0.1g的硫酸铵、2.5g的棉籽饼粉、0.2g的七水硫酸镁，混合充分溶解后用NaOH调节pH为7.0；种子培养每瓶分装25mL种子培养基；121℃灭菌30min。
(b)发酵培养基：每100mL蒸馏水中加入8g的葡萄糖、0.5g的酵母粉、1g的蛋白粉、2g的棉籽饼粉、0.2g的硫酸铵、0.2g的磷酸氢二钾、0.4g的柠檬酸三钠、0.3g的碳酸钙、5g的菜籽油；混合充分溶解后用NaOH调节pH为7.0；121℃灭菌30min；发酵培养每瓶分装25mL发酵培养基；注：碳酸钙分装到每瓶。
(B)产多杀菌素菌株的筛选
取1mL发酵液，其中加入2mL无水乙醇，水浴超声30min后，3500rpm离心10min，取上清用孔径为0.22μm的滤膜过滤后收集滤液。让滤液在流速为1ml/min的条件下，通过C18反相高效液相色谱柱(5μm,250ⅹ4.6mm Agilent,USA)，使用溶剂体系为乙腈:甲醇:0.05％乙酸铵缓冲液＝4.5：4.5：1的溶剂作为洗脱液，在25℃下进行洗脱，使用紫外检测器在波长250nm处进行紫外光吸收检测，收集洗脱液。采用高效液相色谱法(Thermo Fisher)测定了刺糖多孢菌中多杀菌素的产量，并与标准品(含有多杀A和多杀D组分)进行比较，分析发酵产物中是否含有多杀菌素。发现在第3瓶和第21瓶中有多杀菌素产生，将这两瓶的20株菌按照上述(3)中发酵方法进行逐一发酵，最终发现3株可以产生多杀菌素的菌株，产量分别为33.8mg/L，121.5mg/L，161.2mg/L，将其分别命名为LK-001、LK-002和LK-003。
2、高产多杀菌素的菌株的获取
将LK-003的菌株斜面用生理盐水制备成孢子悬液，将其用脱脂棉过滤后调整孢子浓度为10 5～10 6个/mL，取单孢子悬液10mL于无菌的直径9cm平皿内，进行第一轮诱变，具体为在距15W紫外灯20cm处照射30s，然后按照递减10倍进行逐级稀释涂板。
将每20株菌分为一组，按照上述(A)中培养方法进行发酵培养，并通过上述(B)中筛选方法进行菌株筛选，共筛选约3000株菌，将其中产量最高的1组，共20株选为初步筛选菌株， 命名为W100。将这20株菌株进行混合，进行第二轮诱变，第二轮诱变采用化学诱变的方法，即调整孢子浓度为10 5～10 6个/mL，取1mL于一新的离心管中，加入N-甲基-N’硝基-N-亚硝基胍(MNNG)溶液，使诱变浓度为1mg/mL MNNG，共孵育半小时后，用无菌水反复洗涤三次孢子后进行梯度稀释涂板。按此方法筛选约3000株，每20株分为一组进行混合发酵，将其中产量最高的1组，共20株选为第二次筛选菌株，命名为W200。反复进行6轮诱变(3轮紫外诱变，3轮化学诱变，紫外诱变和化学诱变交替进行)后，选最高的一组20株为待筛选菌株，命名为W600。
将最后一轮得到的被命名为W600的20株待筛选菌株进行逐一发酵，参考上述检测方法，在发酵液中均能检测到多杀菌素A和多杀菌素D(图1)，此外还能检测到多杀菌素A-iso、多杀菌素B、多杀菌素E、多杀菌素H、多杀菌素J、多杀菌素K、多杀菌素L、多杀菌素L2、多杀菌素L3、多杀菌素P、多杀菌素P-CH 2(图2)，其中最高产量的菌株为第18株(图3)，即命名为W618，多杀菌素A和多杀菌素D的摇瓶产量总和约为6g/L。
3.菌株W618的鉴定
(1)16srDNA鉴定
通过PCR扩增菌株W618的16SrRNA基因序列，测序，获得其16s rRNA基因的核苷酸序列，具体序列如SEQ ID NO:1所示。经Blast比对，W618的16S rRNA基因与GENBANK ACCESSION CP061007.1的序列相似性最高，相似性为99.93％。根据以上结果，鉴定菌株W618属于刺糖多孢菌(Saccharopolyspora spinosa)。
(2)菌的形态
刺糖多孢菌是好氧型革兰氏阳性放线菌，在大多数培养基(如YMS、苹果酸钙等)上都能生长良好，形成气生菌丝体和白色孢子。菌体适宜的生长温度为25℃～35℃。
菌株W618的形态如图4所示。
综合上述鉴定结果，本发明菌株为刺糖多孢菌(Saccharopolyspora spinosa)。
该菌株W618已于2021年10月12日保藏于中国典型培养物保藏中心(简称CCTCC，地址为中国，武汉，武汉大学)，保藏号为CCTCC NO:M 20211261，分类命名为刺糖多孢菌(Saccharopolyspora spinosa)。
实施例2：高产刺糖多孢菌和野生型刺糖多孢菌菌株产多杀菌素能力比较
1、发酵培养野生型刺糖多孢菌菌株
获取刺糖多孢菌的野生型菌株NRRL18395(美国农业研究菌种保藏中心)，按照实施例1中的发酵方法对野生型刺糖多孢菌进行发酵培养。
2、检测野生型刺糖多孢菌NRRL18395的产多杀菌素能力
参考上述检测方法，分析发酵产物中是否含有多杀菌素，并检测发酵产物中多杀菌素的产量为83.5mg/L。
3、刺糖多孢菌W618与野生型刺糖多孢菌NRRL18395的产多杀菌素能力对比
如实施例1所述，利用本公开提供的刺糖多孢菌W618发酵制备多杀菌素产量高达约6g/L。而野生型刺糖多孢菌产量为83.5mg/L，上述结果说明，本公开筛选获取的W618的产多杀菌素能力显著高于野生型刺糖多孢菌。因此，刺糖多孢菌W618可作为多杀菌素高产的工业化菌株进行开发。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种高产多杀菌素菌株，其保藏号为CCTCC NO:M 20211261。
  2. 一种杀虫组合物，所述组合物包含权利要求1所述的菌株。
  3. 根据权利要求2所述的杀虫组合物，所述组合物还包含选自以下一种或多种化合物：有机磷化合物、氨基甲酸酯类化合物、合成除虫菊酯类化合物、酰基脲化合物、其他类型的昆虫生长调节剂和昆虫激素类似物、类新烟碱和其他烟碱类化合物、大环内酯化合物、其他杀昆虫、杀螨、杀软体动物与杀线虫的化合物或活性成分和吞噬刺激剂；
    优选地，所述有机磷化合物选自以下一种或多种化合物：高灭磷、谷硫磷、硫线磷、壤虫氯磷、毒死蜱、蝇毒磷、dematon、内吸磷、二嗪农、敌敌畏、乐果、EPN、erthoate、灭克磷、氧嘧啶磷、克线磷、杀螟松、丰索磷、倍硫磷、地虫磷、安果、噻唑酮磷、蚜螨磷(庚虫磷)、马来硫磷、甲胺磷、甲基对硫磷(甲基一六0五)、速灭磷、久效磷、对硫磷(一六0五)、甲拌磷、伏杀磷、亚胺硫磷、磷胺、乙丙磷威、肟硫磷(腈肟磷)、丙溴磷、丙虫磷、烯虫磷、丙硫磷、甲基虫螨磷、乙基虫螨磷、喹硫磷(喹噁磷)、硫灭克磷(乙丙硫磷)、嘧丙磷、双硫磷、特丁磷、杀虫畏、thiafenox、二甲硫吸磷(甲基乙拌磷)、三唑磷和敌百虫；
    优选地，所述氨基甲酸酯类化合物选自以下一种或多种化合物：涕灭威、噁虫威、丙硫克百威、杀虫磺、BPMC、氧丁叉威(butoxycarbocim)、甲萘威(西维因)、呋喃丹(虫螨威)、丁硫克百威、除线威、苯虫威、丁苯威、呋线威、灭虫威、异丙威、灭多虫、甲氨叉威、抗蚜威、猛杀威、残杀威、硫双灭多威和thiofurox；
    优选地，所述合成除虫菊酯类化合物选自以下一种或多种化合物：氟酯菊酯、丙烯菊酯、β-氟氯氰菊酯、氟氯菊酯、右旋反灭虫菊酯、氟氯氰菊酯、格林奈、λ-格林奈、γ-格林奈、氯氰菊酯、α-氯氰菊酯、ζ-氯氰菊酯、溴氰菊酯、高氰戊菊酯、杀灭菊酯、五氟苯菊酯、甲氰菊酯、氟氰戊菊酯、氟氯苯菊酯、氯氟胺氰戊菊酯、τ-氯氟胺氰戊菊酯、卤醚菊酯、氯菊酯、protrifenbute、灭虫菊、灭虫硅醚、七氟菊酯、胺菊酯、四溴菊酯、对鱼类安全的类除虫菊酯，例如醚菊酯、天然除虫菊酯类、胺菊酯、S-反丙烯除虫菊、五氟苯菊酯和炔酮菊酯；
    优选地，所述酰基脲、其他类型的昆虫生长调节剂和昆虫激素类似物选自以下一种或多种化合物：噻嗪酮、chromfenozide、定虫隆、伏虫脲(氟脲杀)、双氧威、氟虫脲、特丁苯酰肼、氟铃脲、蒙五一二、氟丙氧脲、蒙五一五、甲氧苯酰肼、双苯氟脲、蚊蝇醚、伏虫隆和双苯酰肼、N-[3，5-二氯-2-氟-4-(1，1，2，3，3，3-六氟丙氧基)苯基]-N’-(2，6-二氟苯甲酰基)脲；
    优选地，所述类新烟碱和其他烟碱类选自以下一种或多种化合物：吡虫清、AKD-1022、巴丹、TI-435、clothiamidin、MTI-446、dinotefuran、吡虫啉、烟碱、硝胺烯啶、thiamethoxam、thiacloprid；
    优选地，所述大环内酯类化合物选自以下一种或多种化合物：阿凡曼菌素、米尔倍霉素、阿巴美丁、伊维菌素、米尔倍霉素和emamectin benzoate；
    优选地，所述其他杀昆虫、杀螨、杀软体动物与杀线虫的化合物或活性成分选自以下一种或多种化合物：阿耳德林、虫螨脒、艾扎丁、唑环锡、联苯肼酯、溴螨酯、氯苯甲脒、氯唑虫清、chlofentezine、乙酯杀螨醇、氯丹、三环锡、灭蝇胺、DDT、三氯杀螨醇、狄氏剂、DNOC、硫丹、ethoxazole、喹螨醚、杀螨锡、唑螨酯、β-唑螨酯、锐劲特、氟苯亚胺噻唑、噻螨酮、IKI-220、噁二唑虫、林丹、灭虫威、蜗牛敌、甲氧滴滴涕、neem、石油与植物油、哒螨酮、拒嗪酮、嘧胺苯醚、鱼藤酮、S-1812、S-9539、spirodiclofen、硫磺、吡螨胺、三氯杀螨砜、唑蚜威、昆虫活性的植物提取物、含有昆虫活性线虫的制备物、可从枯草芽孢杆菌、苏云金芽孢杆菌、核多角体病毒或其他经过遗传修饰或天然生物得到的制备物，以及增效剂，例如胡椒基丁醚、sesamax、safroxan和十二烷基咪唑；
    优选地，所述吞噬刺激剂选自以下一种或多种化合物：葫芦素、糖和Coax。
  4. 根据权利要求2或3所述的杀虫组合物，其中，所述杀虫组合物可以还包含药学上可接受的载体；
    优选地，所述载体是液体载体或固体载体；
    优选地，所述液体载体选自脂肪酮类(如环己酮)和复合醇类(如2-乙氧基乙醇)和芳香化合物，特别是二甲苯和石油馏分，特别是石油的高沸点的萘类和烯类部分如重芳香石油脑中的一种或多种；
    优选地，所述固体载体选自硅镁土黏土、蒙脱石黏土、硅藻土或纯化的硅酸盐中的一种或多种；
    优选地，所述杀虫组合物的剂型选自可湿性粉剂、可溶性粉剂、乳油、水悬浮剂、可分散油悬浮剂、水乳剂、气雾剂、微乳剂和水分散颗粒剂。
  5. 权利要求2-4任一项所述的杀虫组合物的制备方法，其中，所述方法包括将权利要求1所述的菌株接种到培养基中进行发酵培养来制备所述杀虫组合物；
    优选地，所述杀虫组合物选自多杀菌素A、多杀菌素Aiso、多杀菌素B、多杀菌素C、多杀菌素D、多杀菌素E、多杀菌素F、多杀菌素G、多杀菌素H、多杀菌素J、多杀菌素K、多杀菌素L、多杀菌素L2、多杀菌素L3、多杀菌素P、多杀菌素P-CH 2中的一种或多种；
    优选地，所述杀虫组合物包含多杀菌素A和多杀菌素D。
  6. 根据权利要求5所述的制备方法，其中，所述培养基包括种子培养基和发酵培养基，
    优选地，所述种子培养基包含：0.8-1.2质量份的葡萄糖、0.8-1.2质量份的酵母提取物、0.16-0.24质量份的N-Z amine typeA、1.8-2.4质量份的玉米淀粉、0.08-0.12质量份的硫酸铵、2-3质量份的棉籽饼粉、0.16-0.24质量份的七水硫酸镁、80-120质量份的水；优选地，所述水为蒸馏水；更优选地，所述种子培养基的组成为：1质量份的葡萄糖、1质量份的酵母提取物、0.2质量份的N-Z amine typeA、2质量份的玉米淀粉、0.1质量份的硫酸铵、2.5质量份的棉籽饼粉、0.2质量份的七水硫酸镁，100质量份的水；优选地，所述培养基的pH值为6.8-7.2，优选为7.0；
    优选地，所述发酵培养基包含：6.4-9.6质量份的葡萄糖、0.4-0.6质量份酵母粉、0.8-1.2质量份的蛋白粉、1.6-2.4质量份的棉籽饼粉、0.32-0.48质量份的柠檬酸三钠、0.16-0.24质量份的磷酸氢二钾、0.24-0.36质量份的碳酸钙、0.16-0.24质量份的硫酸铵、4-6质量份的菜籽油、80-120质量份的水；优选地，所述水为蒸馏水；更优选地，所述发酵培养基的组成为：8质量份的葡萄糖、0.5质量份的酵母粉、1质量份的蛋白粉、2质量份的棉籽饼粉、0.4质量份的柠檬酸三钠、0.2质量份的磷酸氢二钾、0.3质量份的碳酸钙、0.2质量份的硫酸铵、5质量份的菜籽油、100质量份的水；优选地，所述培养基的pH值为6.8-7.2，优选为7.0。
  7. 根据权利要求5或6所述的制备方法，其中，所述种子培养基和/或发酵培养基用pH调节剂调节pH；
    优选地，pH调节剂是碱；更优选地，所述碱是氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠中的一种或多种。
  8. 根据权利要求5-7中任一项所述的制备方法，其中，所述发酵的条件为200-300rpm、10-40℃、湿度为50-70％；
    优选地，所述发酵条件为250rpm、28℃、湿度60％。
  9. 根据权利要求2-4中任一项所述的杀虫组合物在制备杀昆虫、杀蜱螨亚纲动物、杀线虫的药物的用途。
  10. 根据权利要求9所述的用途，所述杀昆虫、杀蜱螨亚纲动物、杀线虫的药物可以施用于与农业有关的动物或植物；
    优选地，所述动物选自牛、猪、绵羊、山羊、马或驴；
    优选地，所述植物选自大豆、玉米、甘蔗、油菜、马铃薯、棉花、水稻、咖啡、柑橘、扁桃、茶，
    优选地，所述药物可以为对动物通过肠内给药的药物，例如片剂、胶囊、饮品、灌服、 颗粒剂、糊剂、大丸剂、栓剂形式的药物；
    优选地，对动物通过肠胃外给药的药物，例如通过注射(包括肌内、皮下、静脉内、腹膜内等)、植入、淋浴、浸渍、喷雾、倾倒、沾滴、洗涤或撒粉的形式来进行应用的药物；
    优选地，对植物通过喷雾、倾倒、沾滴、洗涤或撒粉的形式来给药的药物。
PCT/CN2022/140418 2021-12-21 2022-12-20 高产多杀菌素菌株及其用途 WO2023116710A1 (zh)

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CN102676393A (zh) * 2011-12-16 2012-09-19 天津北洋百川生物技术有限公司 产多杀菌素刺糖多孢菌及其培养和应用
CN104974974A (zh) * 2015-07-27 2015-10-14 湖南师范大学 一株刺糖多孢菌多杀菌素高产工程菌株及其应用
CN113444659A (zh) * 2021-06-17 2021-09-28 武汉大学 一株高产多杀菌素的刺糖多孢菌

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* Cited by examiner, † Cited by third party
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CN101928670A (zh) * 2009-09-24 2010-12-29 上海天伟生物制药有限公司 一种抗生素的高产菌株及其制备方法和用途
CN102533551A (zh) * 2010-12-15 2012-07-04 上海天伟生物制药有限公司 一种肽类抗生素的高产菌株及其制备方法和用途
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