WO2023138678A1 - 一种贝莱斯芽孢杆菌及其应用 - Google Patents

一种贝莱斯芽孢杆菌及其应用 Download PDF

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Publication number
WO2023138678A1
WO2023138678A1 PCT/CN2023/073307 CN2023073307W WO2023138678A1 WO 2023138678 A1 WO2023138678 A1 WO 2023138678A1 CN 2023073307 W CN2023073307 W CN 2023073307W WO 2023138678 A1 WO2023138678 A1 WO 2023138678A1
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Prior art keywords
plant
bacillus
combination
plants
rot
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PCT/CN2023/073307
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English (en)
French (fr)
Inventor
王雪
梁锏文
余昕彤
季晓琴
王杰
刘仁燕
王琳
蒋先芝
王科晶
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慕恩（广州）生物科技有限公司
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Priority to CN202380013910.0A priority Critical patent/CN118076725A/zh
Publication of WO2023138678A1 publication Critical patent/WO2023138678A1/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
    • 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
    • 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
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • 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
    • A01N63/22—Bacillus

Definitions

  • the invention relates to the technical field of microorganisms and disease prevention and control, in particular to a Bacillus velesii and its application.
  • the biofilm produced by soil microorganisms helps to enhance the competitiveness of plants in the rhizosphere and the advantages of root colonization. It has been reported that microorganisms compete with plant pathogens by producing siderophores, so that plant organisms can directly use iron ions, thereby indirectly protecting plants from pathogens. Microorganisms can induce the accumulation of reactive oxygen species, antioxidants and phytohormones in plants, reduce the negative impact of pathogens on plants, and induce plants to increase the expression of plant-related defense genes and certain defense-related enzymes to enhance the basic immunity of plants against pathogens. Microorganisms can induce plant systemic resistance through their flagellin, lipopeptide substances, lipopolysaccharide, siderophore, etc. as biological elicitors.
  • microbial agents When applying microbial agents to the soil and acting on the root system of crops, it is necessary to consider whether the soil environment is suitable for microbial colonization, such as acid-base, drought, and saline-alkaline; in addition, it is difficult to apply microbial agents to the aboveground parts of crops, that is, the leaf surface, fruit and other parts. Since the application of the leaf surface relative to the root system environment requires consideration of extreme environments such as high temperature, drought, and ultraviolet rays in the leaf environment, the application of leaf spray and spray application methods also need to be considered. Microorganisms are resistant to high temperature, drought, and oxidation, and colonize the leaf surface of crops to exert biological functions.
  • the physical form of the biologic product is also a very important factor that must be considered.
  • microorganisms to form products they must not only have the ability to remain viable for a long time, but they must also have the ability to survive in the environment and perform their intended function. Therefore, the strains with the above characteristics will have important application prospects.
  • the microbial product of the present invention can promote the survival rate of plant transplanting at the seedling stage, and when used in combination with humic acid products, it can significantly promote the growth of plants and effectively prevent and control plant pathogenic bacteria and fungal diseases; experiments have also confirmed that the microbial product of the present invention is suitable for saline-alkali land and improves the soil environment; it has drought resistance and cold resistance; in addition, it can effectively alleviate phytotoxicity; the microbial product of the present invention has been verified to maintain cell viability for at least 12 months without major loss. In pot and field trials, it has shown efficacy in treating diseases caused by different phytopathogenic fungi and in stimulating plant growth. In field trials, it has also shown efficacy against phytopathogenic bacteria, especially tomato bacterial wilt.
  • the present invention provides a Bacillus velezensis M173 (Bacillus velezensis) or a variant thereof or a progeny thereof, the Bacillus velezensis M173 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.61434.
  • said variant or progeny has a similar or identical function to said Bacillus Velez M173.
  • the variant or progeny of the Bacillus velesi M173 has similar or the same physiological and biochemical characteristics as the Bacillus velesi M173.
  • the Bacillus velesi M173 has one or more functions selected from the group consisting of:
  • (4) can promote the growth of plant organs (for example, roots, stems, leaves);
  • the present invention provides a composition comprising Bacillus Velez or a variant thereof or progeny thereof as described above.
  • Bacillus velesii is used together with any of the following components, it means that the composition is different from any organism found in nature.
  • the Bacillus Velez may be in solid form, such as a dried or freeze-dried culture preparation.
  • the composition further comprises one or more additional biological control agents, one or more chemical drugs, or any combination thereof.
  • the composition further comprises an agriculturally or horticulturally acceptable diluent, filler, solvent, spontaneity promoter, carrier, emulsifier, preservative, dispersant, antifreeze, thickener, adjuvant, or any combination thereof.
  • dispersants and/or emulsifiers include all nonionic, anionic or cationic dispersants conventionally used in the formulation of active agrochemical ingredients. It may be preferred to use nonionic or anionic dispersants, or mixtures of nonionic or anionic dispersants.
  • Particularly suitable nonionic dispersants are ethylene oxide-propylene oxide block polymers, alkylphenol polyglycol ethers and tristyrylphenol polyglycol ethers, and their phosphated or sulfated derivatives.
  • Particularly suitable anionic dispersants are lignosulfonates, polyacrylates and arylsulfonate-formaldehyde condensates.
  • examples of preservatives include, but are not limited to, dichlorophen and benzyl alcohol hemiformal.
  • examples of agriculturally or horticulturally acceptable diluents include simple sugars, polysaccharides, molasses, gums, lignosulfonates, aqueous solutions of glycerin, sorbitol, propylene glycol, water, vegetable oils, and mineral oils; carriers may include solids such as alginic acid beads, durum wheat flour (starch) granules, silica, clay, clay minerals, gelatin, cellulose, cellulose derivatives, calcium chlorite, and talc.
  • the support can be a porous solid such as diatomaceous earth, activated carbon, (eg, animal bone charcoal), peat, vermiculite, lignite, wood chips, and corn cobs.
  • compositions within the present invention can be prepared as: aqueous suspensions; stable liquid suspensions; emulsifiable concentrates; capsules; soluble or wettable powders; flowable); dry flowable; wettable granules; wettable dispersible granules; and the like known to those skilled in the art.
  • the additional biological control agent is selected from: bacteria, fungi (eg, yeast), viruses, insects, nematodes, or any combination thereof.
  • the bacterium is selected from the group consisting of Bacillus, Lactobacillus, Bifidobacterium, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
  • the bacteria of the genus Bacillus are selected from the group consisting of: Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus coagulans, or any combination thereof.
  • the additional biological control agent is Bacillus pumilus with a preservation number of GDMCC No.61962, which is preserved in the Guangdong Microorganism Culture Collection Center.
  • the mixed volume ratio of the Bacillus Velez and the Bacillus pumilus in the composition is 1:5 to 5:1 (eg, 1:5; 1:4; 1:3; 1:2; 1:1; 2:1; 3:1; 4:1; 5:1;).
  • the mixed volume ratio of Bacillus Velez and Bacillus pumilus in the composition is 2:1.
  • the yeast is selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces marxianus, or any combination thereof.
  • the chemical drug is selected from the group consisting of herbicides, insecticides, antibacterial agents (eg, fungi, bacteria), antiviral agents, plant growth regulators, antibiotics, fertilizers, or any combination thereof.
  • the antibacterial agent is selected from propamocarb hydrochloride, carbendazim, fluazinam, chlorothalonil, pentachloronitrobenzene, thiram, aluminum triethronate, mefenaxyl, mefenaxyl fludioxonil, or any combination thereof.
  • the insecticide is selected from thiamethoxam, dinotefuran, cyhalothrin, prophom, or any combination thereof.
  • the fertilizer is selected from the group consisting of humic acid fertilizers (e.g., Genroe) urea, ammonium sulfate, Huanzhitian, Antangkeli, potassium dihydrogen phosphate, or any combination thereof.
  • humic acid fertilizers e.g., Genroe
  • ammonium sulfate e.g., Huanzhitian, Antangkeli
  • potassium dihydrogen phosphate e.g., potassium dihydrogen phosphate
  • the composition is in solid form, liquid form, powder form, or any combination thereof.
  • the present invention provides a biological culture comprising Bacillus Velez M173 or a variant thereof or progeny thereof as described above, or a composition as described above.
  • the biological culture further comprises a solid or liquid medium, or a component of such a medium.
  • the biological culture further comprises living cells of Bacillus velesi M173.
  • the living cells are in the form of spores.
  • the spores are present in a liquid culture medium of Bacillus Velez M173.
  • the biological culture comprises cells of Bacillus velesi M173 and a supernatant of the culture.
  • the medium comprises a carbon source (for example, starch hydrolyzate, corn flour, glucose or sucrose), a nitrogen source (for example, soybean meal, peptone), inorganic salts (for example, disodium hydrogen phosphate, magnesium sulfate, sodium carbonate), trace elements (for example, ZnSO4, MnSO4), or any combination thereof.
  • a carbon source for example, starch hydrolyzate, corn flour, glucose or sucrose
  • a nitrogen source for example, soybean meal, peptone
  • inorganic salts for example, disodium hydrogen phosphate, magnesium sulfate, sodium carbonate
  • trace elements for example, ZnSO4, MnSO4
  • the culture further comprises a derivative of Bacillus velesi M173 or a progeny culture thereof.
  • the derivative or progeny culture is selected from a metabolite (e.g., cyclic dipeptide, cyclic tripeptide, bacillusmycin, surfactin), a hormone (e.g., IP, GA3, IPA, IAA, Me-SA, SA, IBA), an enzyme, a cell structural component, or any combination thereof.
  • a metabolite e.g., cyclic dipeptide, cyclic tripeptide, bacillusmycin, surfactin
  • a hormone e.g., IP, GA3, IPA, IAA, Me-SA, SA, IBA
  • an enzyme e.g., a cell structural component, or any combination thereof.
  • the culture further comprises nutrient-providing components.
  • the nutrient-providing ingredient is selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof.
  • the present invention provides a plant growth promoter, which comprises the above-mentioned Bacillus Veles M173 or its variant or its progeny, or the above-mentioned composition, or the above-mentioned biological culture.
  • the plant growth promoter further comprises one or more additional biological control agents, one or more fertilizers, or any combination thereof.
  • the fertilizer is selected from humic acid fertilizers (eg, Genroe) or amino acid water-soluble fertilizers.
  • the present invention provides a biological control agent for preventing and controlling phytopathogenic bacteria and/or promoting plant growth, which comprises the aforementioned Bacillus veles M173 or its variants or progeny thereof, or the aforementioned composition, or the aforementioned biological culture.
  • the present invention provides a growth substrate for plants, which comprises the aforementioned Bacillus veles M173 or its variants or progeny thereof, or the aforementioned composition, or the aforementioned biological culture, or the aforementioned plant growth promoter, or the aforementioned biological control agent;
  • the growth substrate further comprises sand, soil, an inert granular substrate (eg, vermiculite); or any combination thereof.
  • the present invention provides a kind of pesticide composition, and it comprises Bacillus Velez M173 as described above or its variant or progeny thereof, or composition as described above, or biological culture as described above, or plant growth promoter as described above, or biological control agent as described above.
  • the pesticidal composition further includes one or more biological control agents and/or one or more chemical drugs (eg, pesticides).
  • the present invention provides a plant, plant tissue or plant organ treated with the aforementioned Bacillus Velez M173 or its variant or its progeny, or the aforementioned composition, or the aforementioned biological culture, or the aforementioned plant growth promoter, or the aforementioned biological control agent, or the aforementioned pesticide composition.
  • the treating comprises root soaking, foliar spraying, spraying, composting, seed soaking, coating, field irrigation, drip irrigation of plants or plant parts, smearing of plants or plant parts, dripping of plants or plant parts.
  • the plant organ comprises roots, stems, leaves, flowers, fruits and seeds.
  • the plant tissue comprises meristem, protective tissue, basal tissue, and transductive tissue.
  • the plant is selected from the family Solanaceae, Poaceae, Fabaceae, Cucurbitaceae, Brassicaceae, Compositae, Umbelliferae, orchidaceae.
  • the plant has one or more characteristics selected from the group consisting of:
  • the Solanaceae plant is selected from tomato, pepper, potato, eggplant, or any combination thereof;
  • the gramineous plant is selected from corn, wheat, rice, sorghum, or any combination thereof;
  • leguminous plants are selected from soybeans, peanuts, or any combination thereof;
  • the Cucurbitaceae plant is selected from cucumber, wax gourd, pumpkin, bitter gourd, loofah, watermelon, Luo Han Guo, or any combination thereof;
  • the cruciferous plant is selected from Chinese cabbage, rapeseed, cabbage, radish, cauliflower, or any combination thereof;
  • the orchidaceous plant is selected from orchids.
  • the treating includes root irrigation, seed soaking, and seed coating.
  • the present invention provides a method for improving the survival rate of plants at the seedling stage (for example, transplanting at the slow seedling stage), the method comprising: treating the plant with the aforementioned Bacillus velei M173 or its variant or its progeny, or the aforementioned composition, or the aforementioned biological culture, or the aforementioned plant growth promoter or the aforementioned biological control agent or the aforementioned pesticide composition.
  • the treatment is selected from root irrigation, root soaking, foliar spraying, spraying, composting, seed soaking, seed coating, flood irrigation, drip irrigation.
  • the Bacillus Velez M173 improves plant seedling survival rate by one or more characteristics selected from the following:
  • the stress condition is selected from low temperature, drought, high salinity, phytotoxicity (eg, chemical drugs), soil compaction, acidic soil, alkaline soil, high temperature environment, insufficient soil fertility, or any combination thereof.
  • the pathogenic bacteria are selected from pathogenic bacteria, pathogenic fungi or viruses.
  • the pathogen is selected from the group consisting of anthracnose (Colletotrichum capsici) Botrytis cinerea (Botrytis cinerea), ⁇ (Ralstonia solanacearum) ⁇ (Rhizoctonia solani) ⁇ (Fusarium graminearum) ⁇ (Fusarium oxysporum) ⁇ (Athelia rolfsii) ⁇ (Streptomyces scabies) ⁇ (Sclerotium rolfsii) ⁇ (Sclerotiniasclerotiorum) ⁇ (Fusariumoxysporum.sp.cucumebrium Owen) ⁇ (Gaeumannomycescritici) ⁇ (Fusariumgraminearum) ⁇ (Valsamali) ⁇ (Glomerellacingulata) ⁇ (Rhizoctoniasolan) ⁇ (Pyriculariagrisea) ⁇ (Alternariasolani) ⁇ (Exserohilumturcicum) ⁇ (Bipolaria
  • the plant disease caused by the pathogen is selected from bacterial wilt of plants (for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt), soft rot of plants (for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot); plant stalk rot (for example, corn stalk rot, tomato stalk rot); gray mold of plants (for example, botrytis cinerea).
  • bacterial wilt of plants for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt
  • soft rot of plants for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot
  • plant stalk rot for example, corn stalk rot, tomato stalk rot
  • gray mold of plants for example, botrytis cinerea
  • the present invention provides a use of the aforementioned Bacillus Velez M173 or its variant or its progeny, or the aforementioned composition, or the aforementioned biological culture, or the aforementioned plant growth promoter, or the aforementioned biological control agent, or the aforementioned pesticide composition in the prevention and/or treatment of plant diseases caused by pathogenic bacteria, or in the prevention and/or mitigation of plant damage or necrosis caused by non-pathogenic bacteria.
  • the pathogenic bacteria are selected from pathogenic bacteria, pathogenic fungi or viruses.
  • the pathogen is selected from the group consisting of anthracnose (Colletotrichum capsici) gray mold (Botrytis cinerea), Ralstonia solanacearum (Ralstonia solanacearum), Rhizoctonia solani (Rhizoctonia solani) ⁇ (Fusarium graminearum) ⁇ (Fusarium oxysporum) ⁇ (Athelia rolfsii) ⁇ (Streptomyces scabies) ⁇ (Sclerotium rolfsii) ⁇ (Sclerotiniasclerotiorum) ⁇ (Fusariumoxysporum.sp.cucumebrium Owen) ⁇ (Gaeumannomycescritici) ⁇ (Fusariumgraminearum) ⁇ (Valsamali) ⁇ (Glomerellacingulata) ⁇ (Rhizoctoniasolan) ⁇ (Pyriculariagrisea) ⁇ (Alternariasolani) ⁇ (Exserohilumtur
  • the plant disease caused by the pathogen is selected from bacterial wilt of plants (for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt), soft rot of plants (for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot); plant stalk rot (for example, corn stalk rot, tomato stalk rot); gray mold of plants (for example, botrytis cinerea).
  • bacterial wilt of plants for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt
  • soft rot of plants for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot
  • plant stalk rot for example, corn stalk rot, tomato stalk rot
  • gray mold of plants for example, botrytis cinerea
  • the non-pathogenic bacteria condition is selected from low temperature, drought, high salinity, phytotoxicity (eg, chemical drugs), soil compaction, acidic soil, alkaline soil, high temperature environment, insufficient soil fertility, or any combination thereof.
  • the plant is selected from the family Solanaceae, Poaceae, Fabaceae, Cucurbitaceae, Brassicaceae, Compositae, Umbelliferae, orchidaceae.
  • the plant has one or more characteristics selected from the group consisting of:
  • the Solanaceae plant is selected from tomato, pepper, potato, eggplant, or any combination thereof;
  • the gramineous plant is selected from corn, wheat, rice, sorghum, or any combination thereof;
  • leguminous plants are selected from soybeans, peanuts, or any combination thereof;
  • the Cucurbitaceae plant is selected from cucumber, wax gourd, pumpkin, bitter gourd, sponge gourd, watermelon, Luo Han Guo, or any combination thereof;
  • the cruciferous plant is selected from Chinese cabbage, rapeseed, cabbage, radish, cauliflower, or any combination thereof;
  • the orchidaceous plant is selected from orchids.
  • the present invention provides a kind of Bacillus Velez M173 as described above or its variant or progeny thereof, or the composition as described above, or the biological culture as described above, or the plant growth promoter as described above, or the biological control agent as described above, or the use of the pesticide composition as described above in improving the resistance of plants to pathogenic bacteria or adversity conditions, or in promoting plant growth.
  • the pathogenic bacteria are selected from pathogenic bacteria, pathogenic fungi or viruses.
  • the pathogen is selected from the group consisting of Colletotrichum capsici, Botrytis cinerea, Ralstonia solanacearum, Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum, Athena lia rolfsii), Streptomyces scabies, Sclerotium rolfsii, Sclerotias clerotiorum, Fusarium oxysporum.sp.
  • the plant disease caused by the pathogen is selected from bacterial wilt of plants (for example, bacterial wilt of Solanaceae, for example, bacterial wilt of pepper, bacterial wilt of tomato), soft rot of plants (for example, soft rot of grass Rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot); stalk rot of plants (for example, corn stalk rot, tomato stalk rot); gray mold of plants (for example, Botrytis cinerea).
  • bacterial wilt of plants for example, bacterial wilt of Solanaceae, for example, bacterial wilt of pepper, bacterial wilt of tomato
  • soft rot of plants for example, soft rot of grass Rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot
  • stalk rot of plants for example, corn stalk rot, tomato stalk rot
  • gray mold of plants for example, Botrytis cinerea
  • the stress condition is selected from low temperature, drought, high salinity, phytotoxicity (eg, chemical drugs), soil compaction, acidic soil, alkaline soil, high temperature environment, insufficient soil fertility, or any combination thereof.
  • the plant is selected from the family Solanaceae, Poaceae, Fabaceae, Cucurbitaceae, Brassicaceae, Compositae, Umbelliferae, orchidaceae.
  • the plant has one or more characteristics selected from the group consisting of:
  • the Solanaceae plant is selected from tomato, pepper, potato, eggplant, or any combination thereof;
  • the gramineous plant is selected from corn, wheat, rice, sorghum, or any combination thereof;
  • leguminous plants are selected from soybeans, peanuts, or any combination thereof;
  • the Cucurbitaceae plant is selected from cucumber, wax gourd, pumpkin, bitter gourd, loofah, watermelon, Luo Han Guo, or any combination thereof;
  • the cruciferous plant is selected from Chinese cabbage, rapeseed, cabbage, radish, cauliflower, or any combination thereof;
  • the orchidaceous plant is selected from orchids.
  • the present invention provides a method for preventing and/or treating plant diseases caused by pathogenic bacteria, or preventing and/or alleviating plant damage or necrosis caused by non-pathogenic bacteria conditions, said method comprising: applying the aforementioned Bacillus Bacillus M173 or its variant or its progeny, or the aforementioned composition, or the aforementioned biological culture, or the aforementioned plant growth promoter, or the aforementioned biological control agent, or the aforementioned pesticide composition to plants, plant tissues or plant organs.
  • the administration method of said Bacillus veleisi and its composition is familiar to those of ordinary skill.
  • the objects of these methods may be seeds, seedlings, plants, crops, plant parts, flowers, fruits, plant vegetative parts (such as seed tubers and plant cuttings), soils and artificial substrate systems for cultivating plant material.
  • the applying comprises root dipping, foliar spraying, spraying, composting, seed soaking, coating, field irrigation, drip irrigation of plants or plant parts, smearing of plants or plant parts, dripping of plants or plant parts.
  • the plant organ comprises roots, stems, leaves, flowers, fruits and seeds.
  • the plant tissues comprise meristems, protective tissues, basal tissues, and leading organization.
  • the pathogenic bacteria are selected from pathogenic bacteria, pathogenic fungi or viruses.
  • the pathogen is selected from the group consisting of Colletotrichum capsici, Botrytis cinerea, Ralstonia solanacearum, Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum, Athena lia rolfsii), Streptomyces scabies, Sclerotium rolfsii, Sclerotias clerotiorum, Fusarium oxysporum.sp.
  • the plant disease caused by the pathogen is selected from bacterial wilt of plants (for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt), soft rot of plants (for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot); plant stalk rot (for example, corn stalk rot, tomato stalk rot); gray mold of plants (for example, botrytis cinerea).
  • bacterial wilt of plants for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt
  • soft rot of plants for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot
  • plant stalk rot for example, corn stalk rot, tomato stalk rot
  • gray mold of plants for example, botrytis cinerea
  • the non-pathogenic bacteria condition is selected from low temperature, drought, high salinity, phytotoxicity (eg, chemical drugs), soil compaction, acidic soil, alkaline soil, high temperature environment, insufficient soil fertility, or any combination thereof.
  • the plant is selected from the family Solanaceae, Poaceae, Fabaceae, Cucurbitaceae, Brassicaceae, Compositae, Umbelliferae, orchidaceae.
  • the plant has one or more characteristics selected from the group consisting of:
  • the Solanaceae plant is selected from tomato, pepper, potato, eggplant, or any combination thereof;
  • the gramineous plant is selected from corn, wheat, rice, sorghum, or any combination thereof;
  • leguminous plants are selected from soybeans, peanuts, or any combination thereof;
  • the Cucurbitaceae plant is selected from cucumber, wax gourd, pumpkin, bitter gourd, loofah, watermelon, Luo Han Guo, or any combination thereof;
  • the cruciferous plant is selected from Chinese cabbage, rapeseed, cabbage, radish, cauliflower, or any combination thereof;
  • the orchidaceous plant is selected from orchids.
  • the present invention provides a method for improving the resistance of plants to pathogenic bacteria or adversity conditions, or promoting plant growth, said method comprising: applying the aforementioned Bacillus Velez M173 or its variant or its progeny, or the aforementioned composition, or the aforementioned biological culture, or the aforementioned plant growth promoter, or the aforementioned biological control agent, or the aforementioned pesticide composition to plants, plant tissues or plant organs.
  • the administration method of said Bacillus veleisi and its composition is familiar to those of ordinary skill.
  • the objects of these methods may be seeds, seedlings, plants, crops, plant parts, flowers, fruits, plant vegetative parts (such as seed tubers and plant cuttings), soils and artificial substrate systems for cultivating plant material.
  • the applying comprises root dipping, foliar spraying, spraying, composting, seed soaking, coating, field irrigation, drip irrigation of plants or plant parts, smearing of plants or plant parts, dripping of plants or plant parts.
  • the plant organ comprises roots, stems, leaves, flowers, fruits and seeds.
  • the plant tissue comprises meristem, protective tissue, basal tissue, and transductive tissue.
  • the pathogenic bacteria are selected from pathogenic bacteria, pathogenic fungi or viruses.
  • the pathogen is selected from the group consisting of Colletotrichum capsici, Botrytis cinerea, Ralstonia solanacearum, Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum, Athelia ro lfsii), Streptomyces scabies, Sclerotium rolfsii, Sclerotias clerotiorum, Fusarium oxysporum.sp.cucumebrium Owen, Take-all of wheat (Gaeumannomycescritici) ⁇ (Fusariumgraminearum) ⁇ (Valsamali) ⁇ (Glomerellacingulata) ⁇ (Rhizoctoniasolan) ⁇ (Pyriculariagrisea) ⁇ (Alternariasolani) ⁇ (Exserohilumturcicum) ⁇ (Bipolariamaydis) ⁇ (Phytophthoracapsici) ⁇ (Phytophthorani
  • the plant disease caused by the pathogen is selected from bacterial wilt of plants (for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt), soft rot of plants (for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot); plant stalk rot (for example, corn stalk rot, tomato stalk rot); gray mold of plants (for example, botrytis cinerea).
  • bacterial wilt of plants for example, Solanaceae bacterial wilt, for example, pepper bacterial wilt, tomato bacterial wilt
  • soft rot of plants for example, Gramineae soft rot, Orchidaceae soft rot; for example, corn soft rot, orchid soft rot
  • plant stalk rot for example, corn stalk rot, tomato stalk rot
  • gray mold of plants for example, botrytis cinerea
  • the stress condition is selected from low temperature, drought, high salinity, phytotoxicity (eg, chemical drugs), soil compaction, acidic soil, alkaline soil, high temperature environment, insufficient soil fertility, or any combination thereof.
  • the plant is selected from the family Solanaceae, Poaceae, Fabaceae, Cucurbitaceae, Brassicaceae, Compositae, Umbelliferae, orchidaceae.
  • the plant has one or more characteristics selected from the group consisting of:
  • the Solanaceae plant is selected from tomato, pepper, potato, eggplant, or any combination thereof;
  • the gramineous plant is selected from corn, wheat, rice, sorghum, or any combination thereof;
  • leguminous plants are selected from soybeans, peanuts, or any combination thereof;
  • the Cucurbitaceae plant is selected from cucumber, wax gourd, pumpkin, bitter gourd, loofah, watermelon, Luo Han Guo, or any combination thereof;
  • the cruciferous plant is selected from Chinese cabbage, rapeseed, cabbage, radish, cauliflower, or any combination thereof;
  • the orchidaceous plant is selected from orchids.
  • the present application provides a method for inhibiting bacterial wilt disease of plants, the method comprising: preparing the aforementioned Bacillus velei M173 or its variants or progeny thereof into a biopesticide formulation; applying an effective amount of the biopesticide formulation to plants or parts thereof that are infected by R. solanacearum or at risk of being infected by R. solanacearum; in certain embodiments, the plants are tomatoes or peppers.
  • the biopesticide formulation is applied by spraying, foliar spraying, root soaking, or root irrigation.
  • biological control refers to a means of controlling a pathogen by another organism.
  • biocontrol agent refers to an organism (eg, a microorganism) capable of effecting biological control.
  • biocontrol agents include not only microorganisms that are resistant to pathogenic bacteria, but also microorganisms that have beneficial effects on plant health, growth, vigor, stress response or yield.
  • Routes of application of biocontrol agents include spray application, soil application (eg, root irrigation), and seed treatment (eg, seed coating).
  • the purpose of the present invention is to provide a bacterial composition and its application, and the Bacillus velei provided by the present invention is a commercially available biocontrol product, easy to cultivate and suitable for industrial production.
  • the invention provides an application of a fungal agent composition in the prevention and treatment of the following diseases, the diseases are selected from at least one of the following diseases: plant bacterial diseases, plant fungal diseases, plant soil-borne diseases and plant oomycete diseases;
  • the bacterial agent composition includes: Bacillus Veles and Bacillus pumilus M101;
  • Bacterial strain M173 of the species Bacillus velez was deposited in the Guangdong Microbial Culture Collection Center on January 15, 2021, with the preservation number: GDMCC: No.61434;
  • the Bacillus Velez provided by the present invention is isolated from agricultural soil collected from a tobacco plantation site in Yuxi City, Yunnan province (39°54′39.33′′ north latitude, 116°24′48.18′′ east longitude). Deposited in Guangdong on January 15, 2021 Institution Microbial Culture Collection Center, the preservation number is: GDMCC No.61434, and the preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City.
  • the isolated strain is a spore-forming Gram-positive bacterium, which was inoculated in Luria-Bertani medium and cultured at 30°C for 1 day. Its colonies are mostly white, with rough surface and edges, sticky.
  • the sequence of the strain was determined for 16S sequence fragments (amplification primers and sequencing primers are both 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the measurement results were shown in SEQ ID NO.1.
  • amplification primers and sequencing primers are both 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'
  • GDMCC Guangdong Microbial Culture Collection Center
  • the isolated strains were spread on the R2A solid medium and cultured in a 30°C constant temperature incubator. After 1 day, a single colony was picked, continued to inoculate on the R2A solid medium, and cultured upside down in a 30°C constant temperature incubator for 1 day. Then observe the colony morphology, and observe the bacterial cell morphology with an optical microscope (1000 ⁇ ).
  • the strain is Gram-positive, and the colonies are opaque milky white and round in shape.
  • the strains are rod-shaped, round-ended, single or arranged in short chains, about 2.0 microns long. Can move, spore 1.0 ⁇ 1.2 ⁇ 1.5 ⁇ 2.0 microns, oval.
  • the sequence of the strain was determined by 16S sequence fragment (amplification primer and sequencing primer: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3').
  • the measurement results are shown in the sequence table SEQ ID NO.2, 16SrRNA sequence homology analysis, phylogenetic analysis, and through BLAST homologous comparison, we determined that the nearest species of the strain is Pumilis Bacillus pumilus, and named Bacillus pumilus M101.
  • the Bacillus Velez provided by the invention can effectively and stably realize the prevention and treatment of plant bacterial diseases, plant fungal diseases, plant soil-borne diseases and/or plant oomycete diseases, and has strong ability to form biofilms and is easy to colonize. It is suitable for industrialized production and has great prospects for commercialization.
  • the Bacillus Velez provided by the invention can increase the germination rate of seeds in a soil environment with a large number of pathogenic bacteria, and has a good growth-promoting effect.
  • the above-mentioned plant soil-borne diseases include but are not limited to Fusarium oxysporum f.sp. , Fusarium oxysporum.sp.cucumebrium Owen, Gaeumannomycescritici, Fusarium graminearum, Valsamali, Glomerellacingulata, Rhizoctonia solan, Pyriculariag risea), Alternaria solani, Exserohilum turcicum, Bipolaria maydis, Phytophthora capsici and/or Phytophthoranicotianae.
  • the Fusarium is Fusarium graminearum.
  • the application temperature is 2-40°C; even in an optional embodiment, the application temperature is 20-35°C.
  • the above-mentioned plant bacterial diseases include but are not limited to Raueria solanacearum, (Bacillus subtilis), kiwi fruit canker (Pseudomonas syringae), rice bacterial blight (Xanthomonas campestris), cabbage soft rot (Erwinia cartorora), walnut black spot fungus (Xanthomonas campestris), and/or konjac soft rot Bacterial diseases caused by (Erwinia carotovora);
  • the application temperature is 2-40°C; even in an optional embodiment, the application temperature is 20-35°C.
  • the above-mentioned plant fungal diseases or plant oomycete diseases include but are not limited to Rhizoctonia solani, Fusarium, Sclerotias clerotiorum, Fusarium oxysporum f.sp.cucurmerimum, Botrytis cirerea, Fusarium oxysporum.sp.cucu mebrium Owen), Gaeumannomycescritici, Fusarium graminearum, Valsamali, Glomerellacingulata, Rice sheath blight (Rhizoctoniasolan), Pyricularia grisea, Alternaria solani, Botrytis cirerea, Phytophthorain festans, Exserohilum turcicum, Bipolaria maydis, Fusarium oxyspor umf.sp.niveum), Verticillium dahliae, Fusarium oxysporum f.sp
  • the Fusarium is Fusarium graminearum.
  • the application temperature is 2-40°C; even in an optional embodiment, the application temperature is 20-35°C. For example 28°C or 30°C.
  • the present invention also provides
  • the bacterial composition includes: Bacillus Velez and Bacillus pumilus M101; the bacterial strain of Bacillus Velez was deposited in the Guangdong Microbial Culture Collection Center on January 15, 2021, and the preservation number is: GDMCC: No.61434;
  • the above-mentioned plants are gramineous crops or commercial crops.
  • grass crops include, but are not limited to, corn, wheat, rice, sorghum, barley, oats, rye, millet, millet, barnyard millet, and buckwheat.
  • the economic crops are selected from at least one of the following economic crops: Solanaceae, Rosaceae, Rutaceae, Musa, Cucurbitaceae, Fabaceae, Compositae, Liliaceae, Zingiberaceae, Passiflora, Bromeliaceae, Araliaceae and Cactus;
  • Solanaceae is selected from at least one of the following Solanaceae crops: potato, pepper and tomato; Rosaceae is selected from at least one of the following Rosaceae crops: strawberry and papaya; Rutaceae is selected from the following Rutaceae crops: citrus; Musaceae is selected from the following Musaceae crops: banana; Araliaceae is selected from Panax notoginseng, and Cactus is selected from dragon fruit.
  • the mixing volume ratio of Bacillus Veles and Bacillus pumilus M101 in the microbial composition is 1-2:1-2.
  • the mixing volume ratio of the Bacillus velesi and the Bacillus pumilus M101 in the bacterial agent composition is 2:1.
  • the compounded composition can significantly reduce the death rate of plants, and has very good biocontrol potential for plant bacterial diseases, plant fungal diseases, plant soil-borne diseases and plant oomycete diseases.
  • the present invention also provides a bacterial agent, which includes Bacillus Velez and Bacillus pumilus M101; the bacterial strain of Bacillus Velez was preserved in the Guangdong Microbial Culture Collection Center on January 15, 2021, and the preservation number is: GDMCC: No.61434;
  • the bacterial agent is a fermentation culture of Bacillus Velez and Bacillus pumilus M101, and the mixed concentration of Bacillus Velez and Bacillus pumilus M101 in the fermentation culture is 10 6 -10 10 CFU/mL.
  • the mixed concentration of Bacillus Velez and Bacillus pumilus M101 in the fermentation culture is 10 6 -10 10 CFU/mL.
  • 10 6 , 10 7 , 10 8 , 10 9 , 10 10 CFU/mL is a fermentation culture of Bacillus Velez and Bacillus pumilus M101
  • the mixed concentration of Bacillus Velez and Bacillus pumilus M101 in the fermentation culture is 10 6 -10 10 CFU/mL.
  • the present invention also provides a seed coating agent, which includes the seed coating agent and the above bacterial agent.
  • the seed coating agent can increase the germination rate of seeds in a soil environment with a large number of pathogenic bacteria, and has a good growth-promoting effect.
  • the mixing volume ratio of the seed coating agent and the bacterial agent is 8-10:1; preferably, the OD 600 value of Bacillus Veles in the bacterial agent is 0.8-10, and the value of the Bacillus pumilus M101 OD 600 in the bacterial agent is 0.8-10.
  • the formulation of the above-mentioned seed coating is as follows: 5% sucrose, 10% PVPK30, 3% PEG, 2% dispersant MF, 5% polyethylene glycol. Each component is proportioned according to the mass fraction.
  • the preparation method is as follows: Take a 50mL centrifuge tube, first add PVPK30 and a small amount of water according to the above ratio, shake to dissolve, and then add other reagents according to the ratio, dissolve completely, and constant volume to obtain the required seed coating agent.
  • the present invention also provides a root-irrigating agent or a seed-soaking agent, which includes the above bacterial agent.
  • a root-irrigating agent or a seed-soaking agent which includes the above bacterial agent.
  • the above-mentioned root irrigation agent can also be a root soaking agent.
  • the seed soaking agent or root irrigation agent refers to the culture solution of Bacillus Veles and Bacillus pumilus M101, or the freeze-dried powder of Bacillus Velez and Bacillus pumilus M101.
  • the above-mentioned seed soaking agent or root irrigation agent can also be a suspension, an emulsion, or a solution of Bacillus Velez and Bacillus pumilus M101.
  • the concentration of Bacillus pumilus in the culture solution is 10 7 -10 8 CFU/mL
  • the concentration of Bacillus pumilus M101 in the culture solution is 10 7 -10 8 CFU/mL.
  • the Bacillus Velez and Bacillus pumilus M101 provided by the present invention can efficiently and stably prevent and control plant bacterial diseases, plant fungal diseases, plant soil-borne diseases and/or plant oomycete diseases, and the biofilm formed by them is strong, easy to colonize, suitable for industrial production, and has great commercialization prospects.
  • the Bacillus Velez and Bacillus pumilus M101 provided by the present invention can increase the germination rate of seeds in a soil environment with a large number of pathogenic bacteria, and have a good growth-promoting effect.
  • Bacillus Velez and Bacillus pumilus M101 provided by the present invention to soak roots to treat plants can not only effectively prevent and control plant diseases, but its control effect is even better than traditional chemical pesticides. It can be used to replace/partially replace traditional chemical pesticides, and has broad market prospects.
  • the Bacillus Velez M173 of the present invention has relatively prominent strain characteristics. Specifically, (1) has a strong ability to secrete siderophores; (2) has a strong ability to form biofilms; (3) has a stable number of viable bacteria and a long shelf life; M173 grows fast and is not prone to autolysis during the fermentation process; (4) can produce a variety of amino acids and hormones to promote plant resistance to pathogens; (5) can produce a series of cyclic peptide compounds to help plants resist fungi, bacteria and disease infection; High safety factor; (8) Compatible with common fungicides, insecticides and chemical fertilizers on the market; (9) Can improve the stress resistance of crops (for example, salt resistance, low temperature resistance, drought resistance); (10) Can alleviate the chemical damage to crops; (11) It has a synergistic effect of promoting growth when used together with humic acid.
  • Bacillus Velez M173 of the present invention after the Bacillus Velez M173 of the present invention is prepared into a seed coating agent, it can promote plant growth and germination, and significantly improve its control effect on pathogenic bacteria. Moreover, applying it to crops can alleviate the deadness of crops at the seedling stage. Particularly, no matter pot experiment or field experiment, all confirmed the present invention Bacillus Velez M173 has significant control effects on fungal and bacterial diseases, especially the control of bacterial wilt and stem rot. Therefore, the Bacillus Velez M173 of the present invention has relatively wide application prospects.
  • Figure 1 is a graph showing the colony morphology of the M173 strain on LB medium.
  • Fig. 2 is a picture of the biocontrol effect of the M173 bacterial agent on bacterial wilt by cutting leaves.
  • Fig. 3 is a picture of the biocontrol effect of the Bacillus velei M173 strain on bacterial wilt.
  • Fig. 4 is a picture of the biocontrol effect of the Bacillus velei M173 strain on Fusarium graminearum.
  • Fig. 5 is the siderophore ability result of Bacillus velaisi M173 strain.
  • Fig. 6 is the growth curve of the Bacillus velei M173 strain and the strains of competing products.
  • Figure 7 is the growth curve of Bacillus Velez M173 and other Bacillus Velez.
  • Fig. 8 is the effect of different pH on the bacterial count of the M173 Bacillus velei M173 strain.
  • Fig. 9 is the effect of different NaCl contents (%) on the growth of the bacterial count of Bacillus Velez M173 strain.
  • Figure 10 shows the growth-promoting effects of different groups of treatments on cucumber.
  • Fig. 11 shows the growth status of cucumber seedlings applied with Bacillus velei M173 strain at different salt concentrations.
  • Fig. 12 shows the situation of cucumbers in the Bacillus veles M173 group and the UTC group (untreated group) after watering with 200 mM sodium chloride.
  • Figure 13 shows the damage of cucumbers under the phenomenon of salt damage.
  • Fig. 14 is the condition of growth of cucumber seedlings in each treatment of antidote damage.
  • Fig. 15 is the growth situation of cucumber seedlings of each treatment in the cold resistance test.
  • Fig. 16 is the growth situation of cucumber seedlings of each treatment in the drought resistance test.
  • Figure 17 is the plate bacteriostasis effect of Bacillus Velez M173.
  • Figure 18 shows the growth of peanut seedlings coated with Bacillus velei M173.
  • Figure 19 is the situation of peanut seeds coated with Bacillus velei M173.
  • Fig. 20 is the plant height of bitter melon treated by different groups.
  • Figure 21 shows the growth and number of fruits of bitter melon after different groups of treatments.
  • Figure 22 is the result of preventing dead trees of Luo Han Guo after different groups of treatments.
  • Fig. 23 is the control effect of different groups on tomato sore wilt after the experimental site 1 is treated.
  • Figure 24 shows the results of different groups of treatments on the control effect and incidence of tomato bacterial wilt in the experimental plot 3.
  • Fig. 25 is the control effect of different groups on tomato sore wilt after the experimental plot 4 is treated.
  • Figure 26 is the incidence rate of orchid stem rot after different groups of treatments in the first disease investigation.
  • Figure 27 is the relative control effect of orchid stem rot after different groups of treatments in the first onset investigation.
  • Figure 28 is the incidence rate of orchid stem rot after different groups of treatments in the second incidence survey.
  • Figure 29 is the relative control effect of orchid stem rot after different groups of treatments in the second outbreak investigation.
  • Figure 30 shows the growth of orchids in the M173 (400-fold dilution) group.
  • Figure 31 shows the growth of orchids in the M173 (200-fold dilution) group.
  • Figure 32 shows the growth of orchids in Zhuorun group.
  • Figure 33 shows the growth of orchids in the 3% metalapam ⁇ hymexazol group.
  • This example provides a screening and identification method for the Bacillus velei M173 strain.
  • Bacillus velezensis M173 was isolated from the agricultural soil collected from the tobacco planting area in Yuxi City, Yunnan province (39°54′39.33′′N, 116°24′48.18′′E).
  • the isolated strains were inoculated in Luria-Bertani medium and cultured at 30°C for 1 day.
  • the morphology is shown in Figure 1.
  • the strain was identified as a spore-forming Gram-positive bacterium, and its colonies were mostly white, with rough surfaces and edges, and sticky.
  • the sequence of the strain was determined for 16S sequence fragments (amplification primers and sequencing primers were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the determination results were shown in SEQ ID NO: 1.
  • Bacillus velezensis M173 Send Bacillus Velez M173 to Guangdong Microbial Culture Collection Center (GDMCC) for preservation.
  • GDMCC Guangdong Microbial Culture Collection Center
  • R2A solid medium Weigh 18.1g R2A agar medium dry powder (Guangdong Huankai Biotechnology Co., Ltd.), add water to 1L, and sterilize by high-pressure steam at 121°C for 20 minutes.
  • the strain is Gram-positive, and the colonies are opaque milky white and round in shape.
  • the strains are rod-shaped, round-ended, single or arranged in short chains, about 2.0 microns long. Can move, spore 1.0 ⁇ 1.2 ⁇ 1.5 ⁇ 2.0 microns, oval.
  • the sequence of the strain was determined for the 16S sequence fragment (amplification primer and sequencing primer: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3').
  • the measurement results are shown in the sequence table SEQ NO: 1, 16SrRNA sequence homology analysis, phylogenetic analysis, and through BLAST homologous comparison, we determined that the closest species of the strain is Bacillus pumilus ( Bacillus pumilus), and named Bacillus pumilus M101.
  • Bacillus pumilus M101 has been sent to the Guangdong Microbial Culture Collection Center (GDMCC) for preservation.
  • the preservation address is: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City.
  • the preservation date is October 22, 2021, and the preservation number is GDMCC No.61962.
  • the fermentation culture method is simple and easy to operate, and is suitable for industrial production.
  • LB solid medium tryptone 10g, yeast powder 5g, NaCl 10g, agar powder 15g, add water to 1L, sterilize by high pressure steam at 121°C for 20 minutes.
  • the M173 strain stored in the -80°C refrigerator was spread on the LB plate by the coating method, and cultured at 30°C for 1 day.
  • the fermentation culture method is simple and easy to operate, and is suitable for industrial production.
  • LB solid medium tryptone 10g, yeast powder 5g, NaCl 10g, agar powder 15g, add water to 1L, sterilize by high pressure steam at 121°C for 20 minutes.
  • the M173 strain stored in the -80°C refrigerator was spread on the LB plate by the coating method, and cultured at 30°C for 1 day.
  • M173, M101 and their compositions were used to verify the leaf-cutting biocontrol effect of the R. solanacearum MN R.s1 strain.
  • R. solanacearum MN R.s1 (Ralstonia solanacearum, stored in Moon Biobank) from a glycerol tube onto a TTC plate (recipe: 5g glucose, 10g peptone, 1g hydrolyzed casein, 15g agar, add water to 1L, sterilize at 121°C for 20min, cool to 60°C and add TTC to make the final concentration 0.005% (W/V)
  • Solanacearum colonies were transferred to SPA liquid medium (formulation: sucrose 20g, bacteriological peptone 5g, K 2 HPO 4 0.5g, MgSO 4 0.25g, add water to 1L, adjust to pH 7.0-7.2, sterilize at 121°C for 20min), culture on a shaker for 8-16h and then according to OD 600
  • the absorbance value of the R.s diluted to 10 7 CFU/mL, the R.s bacterial suspension was prepared for use.
  • a spectrophotometer to measure the absorbance at OD 600 of the fermentation culture of the M101 strain prepared in Example 4, and adjust its concentration to 10 8 CFU/mL with LB liquid medium to obtain the M101 bacterial agent.
  • the leaves were cut to preliminarily judge the biocontrol effect of M173 on R.s.
  • a blank control group, a negative control group, a positive control group and a bacterial agent treatment group were respectively set up, and each group was repeated three times.
  • soak sterile scissors in the soaking solution corresponding to each group for 1 second and then use the soaked scissors to cut off half of the tomato seedling leaves.
  • the tomato seedling pots after leaf clipping were placed in a high-temperature and high-humidity disease shed at 30° C. for 7 days. And from the 4th day after the leaves were cut, the number of dead tomato seedlings was recorded every day, and the mortality rate and relative control effect were calculated.
  • the immersion solution used in each group is as follows:
  • the blank control group was clear water 40mL;
  • Negative control group is 20mL each of clear water and R.s bacteria suspension, mix well;
  • the positive control group is 1000-fold dilution of neophytomycin and 20 mL of Rs bacterial suspension, and mix well;
  • bacterial agent treatment groups There are 5 groups of bacterial agent treatment groups, respectively take 20 mL of bacterial agent prepared in step (3) and step (4) and 20 mL of R.s bacterial suspension, and mix well.
  • Relative control effect (negative control mortality rate-treatment group mortality rate)/negative control mortality rate ⁇ 100%.
  • each seedling of the negative control group, positive control group and bacterial agent treatment group was poured into 50mL of R.s bacteria suspension, and the blank control group was filled with an equal amount of clear water.
  • the disease begins on the 5th day after root irrigation
  • observe and record the state of the tomato seedlings once a day record the disease situation according to the grading standard
  • 10th-13th day determined according to the actual disease situation
  • record the number of plants for each disease level of each treatment and calculate the incidence rate and disease index according to the following formula.
  • Plant disease is divided into the following 5 grades:
  • Disease index (%) ( ⁇ (number of plants in each disease grade ⁇ number of grades)/(total number of plants ⁇ number of highest grades)) ⁇ 100%.
  • Controlling effect (%) ((Illness index of negative control group-Illness index of experimental group)/Illness index of negative control group) ⁇ 100%.
  • Each treatment group of table 2 is to the irrigation root biocontrol effect of bacterial wilt
  • Fusarium graminearum Fg (Fusarium graminearum, stored in Moon Biobank) on a PDA plate, culture at 28°C for 5 days, and then use CMC medium (formulation: CMC-Na 15g, NH 4 NO 3 1g, yeast extract 1g, MgSO 4 7H 2 O 0.5g, KH 2 PO 4 1g, add water to 1L, sterilize at 121°C for 20min) After culturing at 25-28°C and 200 rpm for 7 days, filter the spore suspension with gauze, count it with a hemocytometer, and prepare a spore suspension with a concentration of 10 6 CFU/mL.
  • CMC medium formulation: CMC-Na 15g, NH 4 NO 3 1g, yeast extract 1g, MgSO 4 7H 2 O 0.5g, KH 2 PO 4 1g, add water to 1L, sterilize at 121°C for 20min
  • sucrose 5% sucrose 5%
  • PVPK30 10% 10%
  • PEG 3% polyethylene glycol 5%
  • dispersant MF 2% polyethylene glycol 5%
  • M173 bacterial strain fermentation culture and M101 bacterial strain fermentation culture obtained in Example 3 and Example 4 respectively Measure the absorbance at OD600 of the M173 bacterial strain fermentation culture and M101 bacterial strain fermentation culture obtained in Example 3 and Example 4 respectively with a spectrophotometer, and use LB liquid medium to adjust the OD600 value of the fermentation culture to 1 to prepare M173 bacterial liquid and M101 bacterial liquid. And the obtained M173 bacterial liquid and M101 bacterial liquid are prepared according to the volume ratio of 1:1, 2:1, and 1:2 to prepare the mixed bacterial liquid of M173 and M101 for future use.
  • the wheat seeds (Jimai 23) were coated with the seed-coating agent (400 ⁇ L/20 g of wheat), so that M173 and/or M101 were attached to the surface of the seeds.
  • the seeds of the blank control group were not treated, and the seeds of the negative control group were coated with the control seed coating agent.
  • the germination rate of wheat was counted after germination, and the condition of wheat was counted after 14 days.
  • the classification of the condition is as follows:
  • the disease index of wheat plants in the negative control group was 38.89%
  • the disease index of the M173 treatment group was 20.01%
  • the disease index of the composition 2:1 was only 15.43%
  • its relative control effect was 60.32%. It shows that M173 can effectively control the damage of F.g to wheat plants, and the control effect of the composition 2:1 is better than that of M173.
  • Fusarium graminearum F.g reduced the germination rate of wheat seeds in the negative control group to 77.42%
  • the seed germination rate of the treatment group containing M173 bacterial solution was above 91%
  • the germination rate of wheat seeds in the composition 2:1 was as high as 95%.
  • M173 can effectively ensure the germination rate of plant seeds in the F.g environment, and the composition 2:1 can further increase the germination rate.
  • the presence of the seed coating agent provided by the invention can effectively protect wheat seeds from the influence of Fusarium graminearum F.g, and promote seed germination; and the composition of M173 and M101 has dual effects of further promoting seed germination and efficiently controlling Fusarium graminearum.
  • the biofilm formation ability of the M173, M173 and M101 compositions was further verified to test its colonization ability on field soil/plant leaves, in order to verify the stability and persistence of the control effect of the M173, M173 and M101 compositions.
  • the M173 bacterial strain fermented culture and the M101 bacterial strain fermented culture that embodiment 3 and embodiment 4 gained are measured its absorbance at OD 600 place with spectrophotometer respectively, and fermented culture is fermented with LB liquid medium
  • the OD 600 value of the nutrient was adjusted to 1, and the M173 bacterial liquid and the M101 bacterial liquid were prepared.
  • the obtained M173 bacterial liquid and M101 bacterial liquid are prepared according to the volume ratio of 1:1, 2:1, and 1:2 to prepare the mixed bacterial liquid of M173 and M101 for future use.
  • Irrigate cucumber seedlings with the above bacterial solution 50mL per pot with a total of 6 ⁇ 10 8 cfu, each 6 pots is 1 treatment, and each treatment is repeated 3 times.
  • the control group was irrigated with the same amount of water. After 2 hours of inoculation, two small holes were poked in the opposite positions of each pot, and a 5mm-diameter cake of pathogenic bacteria was connected to each hole, and then placed in a greenhouse at 28-30°C for 7 days.
  • Grade 4 Symptoms such as depression or breakage at the junction of cucumber seedlings, rot and browning, etc.
  • Incidence index ( ⁇ (number of diseased plants at all levels ⁇ representative value of each disease level)/(total number of investigated plants ⁇ highest level of diseased value)) ⁇ 100
  • Relative control effect (%) (disease grade index of control group-disease grade index of treatment group)/disease grade index of control group ⁇ 100%.
  • the cucumber seedling disease grades treated by watering the fermentation culture of composition 2:1 are mainly between 0-1 grades, while the control group cucumber seedling disease grades are mostly in 1-2 grades, and its cucumber seedling disease index (44.59%) is higher than that of the treatment group, and the relative control effect of composition 2:1 to cucumber blight reaches 39.78%. damage to the strain.
  • each treatment and repetition group are randomly arranged.
  • the Institute offers two options, subject to the actual situation.
  • composition provided by the present invention can not only antagonize R. solanacearum, Fusarium graminearum and Rhizoctonia solani, effectively control tomato bacterial wilt, wheat head blight, cucumber blight and wax gourd wilt, but also promote seed germination and increase germination rate; it can be quickly colonized and facilitates industrial production, and has broad application prospects.
  • Siderophores are low-molecular-weight complexes synthesized and secreted by microorganisms that chelate iron for iron intake. Root microorganisms can chelate iron through siderophores and supply them to plants. On the other hand, siderophores produced by biocontrol bacteria can compete with pathogenic bacteria for iron and inhibit the growth and reproduction of pathogenic microorganisms.
  • Iron starvation treatment take a strain of Bacillus pumilus in the Moon bacteria bank as a negative control (for the screening method and identification method of this strain, refer to the content described in Example 2), scrape the test strain with an inoculation loop, inoculate it into a 50 mL centrifuge tube containing 10 mL of MKB iron-free medium, and cultivate it with shaking at 30°C and 200 rpm for 24h-72h. After the cells are produced, centrifuge at 1000rpm for 5min and discard the supernatant. The bacterial cells were washed twice with 5 mL of sterile ultrapure water at 10,000 rpm for 5 min, and then diluted 10 times with sterile ultrapure water to obtain a bacterial suspension.
  • CAS plate covering method Take 10 ⁇ L of bacterial suspension to inoculate on the iron-free MKB plate, or use an inoculation loop to inoculate on the iron-free MKB plate. Three replicates of each strain were placed in a 30°C incubator for 2 days. After 2 days, obvious single colonies grew on each plate. After the CAS medium was cooled to below 60°C, a layer of CAS medium was poured on the plates with colonies, and the color change of each plate was observed after standing for 1 hour. Observe again after 24 hours, and take pictures for records.
  • solubility index that is, the size of the halo ratio D/d
  • Biofilm-forming property refers to the film-forming property of bacteria on the surface of objects after accumulation, and the strains of microbial products with film-forming property are positively correlated with the colonization ability of field application and the effect of disease biocontrol.
  • control strains selected in this example are: Bacillus pumilus M101 identified and obtained in Example 2; competing product 1 - Bacillus subtilis isolated from a commercially available Bacillus subtilis product, and competing product 2 - Bacillus amyloliquefaciens isolated from a commercially available Bacillus amyloliquefaciens product.
  • the 250ml Erlenmeyer flask culture medium loading capacity that has baffle plate is 50mL, cultivates seed liquid (M173 of the present invention and above-mentioned three contrast bacterial strains) to be inoculated into fermentation medium (glucose 5g/L, yeast powder 5g/L, peptone 5g/L, NaCl 5g/L) by 2% (v/v), 30 DEG C, 200rpm cultivates, and regular sampling detects fermented liquid OD600.
  • the experimental results are shown in Figure 6, and the results show that Bacillus Velez M173 has the shortest logarithmic phase, so it can quickly establish a functional flora. Also, Bacillus Velez M173 has a long stable period, so it can produce a large amount of secondary metabolites.
  • M173 can grow rapidly, has a long shelf life, can secrete a large amount of secondary metabolites that inhibit pathogenic bacteria, and directly kill pathogenic bacteria; has a strong ability to form biofilms, quickly forms a protective layer on crop roots, resists pathogenic bacteria invasion, and prevents diseases through dual mechanisms.
  • Bacillus in the later stage of fermentation, when the nutrition is deficient or the conditions become poor, spores will gradually form, but a considerable part of the Bacillus will autolyze and not produce spores, which is a big challenge for large-scale fermentation to form products; the present invention further compares Bacillus Velez M173 with Bacillus Velez 2 and Bacillus Velez 5 recorded in Example 19, comprehensive growth curve, spore-forming situation and autolysis situation. optimal strain;
  • Seed medium LB medium (g/L): peptone 10, yeast powder 5, NaCl 5;
  • Fermentation medium LB medium (g/L): peptone 10, yeast powder 5, NaCl 5;
  • the medium capacity of a 250ml Erlenmeyer flask with baffles is 50mL, prepare 7 bottles of medium for each strain, inoculate the cultured seed solution into the fermentation medium at 2% (v/v), 30°C, 200rpm medium, and take samples at 0h, 4h, 8h, 14h, 18h, 22h, and 24h.
  • E is the activation energy
  • the independent variable can be calculated as temperature T according to the formula, and the K value is further calculated;
  • the K value at room temperature of 25°C is calculated to be 0.000735, and the actual number of bacteria C 0 at the factory is 8.23 ⁇ 10 9 .
  • the half-life t can be calculated to be 943.20. It is predicted that the number of bacteria will remain at 4.11 ⁇ 10 9 cfu 2.5 years after the product leaves the factory (946.20d). /mL, meeting the requirement of product shelf life of 2 years.
  • the number of effective viable bacteria was counted on the plate with a dilution of 20-300 colonies as the counting standard. When there is only one dilution and the average number of colonies is between 20 and 300, the average number of colonies is used for calculation. If there are 2 dilutions and the average number of colonies is between 20 and 300, it should be determined according to the ratio of the total number of colonies between the two. If the ratio is less than or equal to 2, the average of the two should be calculated; if it is greater than 2, the average number of colonies with a small dilution should be used for calculation.
  • the number of viable bacteria per gram of sample (C/V) ⁇ M
  • C is the average number of colonies at a certain dilution concentration
  • V represents the volume (mL) of the dilution solution used when coating the plate
  • M represents the dilution multiple.
  • M173 was a spore-forming bacterium, which formed round or oval spore resting bodies in the bacteria.
  • the spores have extremely low water content, strong stress resistance, and can withstand high temperature, ultraviolet rays, ionizing radiation, and various chemical substances.
  • the index of spore rate is positively correlated with the shelf life of live bacteria products; the test results show that the survival rate of M173 of the present invention can stably reach more than 90%, and the number of spores can reach 7.5 ⁇ 10 9 cfu/mL; the above comprehensive results show that M173 has the characteristics of stable spore number, high number of viable bacteria, and long shelf life. 3 It has the quality characteristics of being listed as a product.
  • M173 Hormone Determination Method 1) Sample preparation: Dissolve M173 sample in 5ml 50% methanol/water, mix evenly, draw 100 ⁇ l sample solution to filter through 0.22 ⁇ m membrane, and finally dilute to 0.5ml in a liquid phase vial. 2) Control: IAA, IBA, methyl jasmonate, gibberellin, methyl salicylate, isopentenyl adenosine growth hormone mixed standard.
  • Test instrument use LC-QQQ (triple quadrupole liquid mass spectrometer) to test the content of growth hormone contained in the sample.
  • Tables 15 and 16 The results of hormone and amino acid assays are shown in Tables 15 and 16.
  • Table 15 shows that the M173 strain can secrete hormones IP, GA3, IPA, IAA, Me-SA, SA, and IBA. It shows that M173 can promote the growth of plants, promote the rooting of plants, and when a part of plants are infected by pathogens, the strain M173 of the present invention can promote the resistance of plants to pathogens, thereby suggesting that it has the use of disease prevention.
  • Table 16 shows that M173 produces a variety of amino acids, and amino acids can improve crop quality, enhance crop metabolism, and promote crop growth.
  • cyclic dipeptides and cyclic tripeptides are compounds with stable structures and various biological activities.
  • Bacillomycin has a strong broad-spectrum inhibitory effect on pathogenic fungi and has the activity of inhibiting pathogenic bacteria.
  • Surfactin is not only antiviral, but also antibacterial and antifungal.
  • Acid and alkali resistance test Prepare LB liquid medium, adjust the pH value of the medium to 3/5/7/9/11 (adjusted with 1mol/L HCl and 1mol/L NaOH), and sterilize under high temperature and high pressure at 121°C for 20min.
  • M173 Activate M173 from glycerol tubes to LB medium solid plate, culture in 30°C incubator for 1-2d, inoculate M173 into LB liquid medium, and culture on shaker for 24h as seed solution, inoculate M173 into LB medium with different pH values according to the inoculum amount of 1mL per bottle, repeat 3 times, culture at constant temperature on shaker at 30°C, and when cultured to 24h and 48h, take samples to measure OD600 value and dilute plate to count the number of viable bacteria.
  • Salt tolerance test Prepare LB medium containing NaCl 0.05mol/L, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L respectively, and sterilize under high temperature and high pressure at 121°C for 20min.
  • the seed solution of M173 was inoculated into LB medium with different salt content according to the inoculum amount of 1 mL per bottle, and the medium without NaCl was used as the control, and the culture medium was repeated three times, and the shaker was kept at a constant temperature of 30°C.
  • M173 can grow in the environment of PH3 ⁇ PH11, can grow in extremely acidic and alkaline environment, and maintain a high number of viable bacteria.
  • M173 can grow and reproduce in various soil environments, especially in strongly acidic soil (pH ⁇ 5.0) and heavily saline-alkali soil (pH>9.5, salt content>0.6%).
  • Seedling cultivation plant 2 tomatoes per pot, 6 pots per plate is 1 treatment, 12 seedlings in total, 3 repetitions per treatment, 36 seedlings in total.
  • Water and fertilizer management In addition to watering the bacteria agent on time, water 0.01% NPK water-soluble fertilizer in case of drought in the middle.
  • Bacteria treatment the 12-day-old seedlings were watered for the first time, and the second watering was performed at intervals of 7 days, totaling 2 times.
  • Bacterial agent dilution Dilute the required bacterial agent to the required multiple with tap water, such as 1L/mu treatment, calculated on the basis of 2000 plants per mu, that is, 0.5mL per plant. Two seedlings in each pot need 1mL of bacterial agent in total, and 50mL of solution is poured, so the dilution factor of this treatment is 50.
  • Plant height the height of the plant (the distance from the root neck to the growing point).
  • Stem thickness measure the stem diameter of the plant based on the first true leaf.
  • M173 of the present invention is safe to tomato, and has a higher safety factor.
  • Example 13 M173 and the growth-promoting ability of M173 combined with humic acid
  • Tested products M173 strain; Genluo (FMC humic acid product); Zhuorun (competitive product).
  • Seedling cultivation Sow 3 cucumber seeds per pot, wait until 12 days to grow to 2 plants per pot, 6 pots per plate is 1 treatment, each treatment is repeated 3 times, a total of 36 seedlings.
  • Water and fertilizer management Water 0.01% NPK water-soluble fertilizer in an appropriate amount to ensure the normal growth of cucumber seedlings.
  • each product was diluted with tap water in different times, and each pot was poured with 50 mL.
  • Spreading degree the distance between the tips of two true leaves of cucumber.
  • Stem thickness measure the stem diameter of the plant based on the first true leaf.
  • the survival rate is calculated by the method of dilution and coating of the bacterial solution, and there may be certain systematic errors in the dilution coating, so the individual survival rate will be greater than 100%; if the survival rate is greater than 100%, it can be understood as M17 3 very good compatibility with chemicals).
  • Embodiment 15.M173 improves crop resistance experiment report
  • Plants are composed of cells, and the stability of cells is mainly maintained by the ion concentration difference between the inside and outside of the cells.
  • the high concentration of Na+ will also hinder the absorption of Ca 2+ by plants, resulting in calcium deficiency in plants, and the phenomenon of seedling failure and fruit failure.
  • the hindered accumulation of calcium ions leads to the obstruction of the absorption of a series of crop elements.
  • the cultivated cucumber seedlings were thinned to 2 seedlings per pot, 6 pots per treatment, and repeated 3 times.
  • two treatments were set up: clear water blank control (UTC) and watering compound bacterial agent.
  • the 12-day-old cucumber seedlings were watered (1 mL per plant, diluted 50 times before watering), and UTC was watered with the same amount of water.
  • Salt stress was carried out after 3 days, and the method of pouring a corresponding concentration of salt solution into the soil matrix was adopted.
  • the overall growth of cucumber seedlings irrigated with M173 was higher than that of the untreated group (UTC) (Table 25 and Figure 11), and their biomass was higher than that of the UTC group.
  • UTC untreated group
  • the dry weight of cucumber showed a downward trend.
  • M173 processing The biomass of cucumber under different salt concentration treatments was higher than that of UTC group, and the growth rate of dry weight was more than 10%. With the increase of salt concentration, the growth rate of cucumber biomass of M173 was higher. Under the condition of 200mM NaCl treatment, the biomass of cucumber treated with M173 increased by 19.19% compared with UTC.
  • M173 can promote the growth of cucumber under normal conditions and salt stress, and M173 can reduce the damage of cucumber caused by high-salt environment. Cucumbers treated with M173 grew better under salt stress and had the effect of reducing salt damage to crops, which provided a theoretical basis for its promotion in unfavorable soil environments with high salt content.
  • Embodiment 16 Antidote damage test pot experiment
  • Nailaxyl Mancozeb is a low-toxic compound fungicide that is mixed with mancozeb and mancozeb in a scientific proportion. It is specially used to prevent and treat low-level fungal diseases. It has dual functions of protection and treatment. The two bactericidal mechanisms complement each other and can delay the development of drug resistance of bacteria. It is safe to use.
  • the cultivated cucumber seedlings were thinned to 2 seedlings per pot, 6 pots per treatment, and repeated 3 times. In the test group, two treatments were set up: clear water blank control (CK) and watering bacterial agents.
  • the cucumber seedlings with true leaves were sprayed with the chemical medicine fine nail cream manganese zinc, and the gradient was set at 500 times (recommended dilution factor), 250 times (2 ⁇ ), and 125 times (4 ⁇ ). Measure each index after 7d. Based on growth data such as plant height, stem diameter, and dry weight, record the level of phytotoxicity at the same time.
  • Level 1 Cucumber seedling leaves are yellowed and the area of spots is less than 1/4
  • Grade 3 Cucumber seedling leaves are yellowed and the spot area is greater than 1/2, but does not cover the entire leaf
  • Phytotoxicity index ( ⁇ (number of leaves at all levels ⁇ representative value at each level)/(total number of leaves under investigation ⁇ highest level of disease incidence)) ⁇ 100
  • Relative control effect (%) (phytotoxicity index of the control group - phytotoxicity index of the treatment group) / phytotoxicity index of the control group ⁇ 100%
  • M173 can alleviate the phytotoxicity of Jingjia Frost ⁇ manganese zinc. Under the condition of high concentration (125 times dilution) chemical spraying, the phytotoxicity control effect of cucumber treated with M173 reached 47.18%.
  • Embodiment 17 Cold resistance test pot experiment
  • M173 can reduce crop growth by promoting crop growth and increasing chlorophyll content. adverse effects of adversity.
  • M173 can promote the growth of cucumber in low temperature environment and reduce the damage caused by low temperature adversity to cucumber.
  • Embodiment 18 Drought resistance test pot experiment
  • PEG-6000 polyethylene glycol
  • PEG-6000 polyethylene glycol
  • the cucumber seedlings about 12 days old were transplanted into non-porous planting pots, 1 seedling per pot, 6 pots of seedlings per treatment, and repeated 3 times.
  • the test group was set up as a clear water blank control (CK-UTC), 20% PEG-6000 treatment (CK-PEG), and watering compound bacterial agents on the basis of PEG treatment. After transplanting, water the compound microbial agent (1mL per plant, dilute it 50 times before watering, it is advisable to water thoroughly), and water the same amount of water for CK-UTC and CK-PEG treatment.
  • M173 can improve the growth of cucumber under drought conditions and reduce the inhibition of drought stress on crop growth, indicating that under drought stress conditions, M173 can reduce the adverse effects of stress by promoting crop growth.
  • Embodiment 19 Flat plate inhibits pathogenic bacteria experiment
  • Botrytis cinerea Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Rhizoctonia solani, Athelia rolfsii, Streptomyces scabies, Ralstonia solan acearum).
  • Blank control group (fungal treatment) fungal cakes (5 mm in diameter) were taken from the edge of fungal pathogen colonies activated for 3 days, placed 2.5 cm away from the center of the PDA plate, and incubated at a constant temperature of 28°C. (Bacteria treatment) Pipette 100 ⁇ L of Streptomyces scabies and R. solanacearum pathogenic bacteria solution onto LA plates, spread evenly and dry in the air.
  • Velez bacillus M173 group (fungistatic test) get bacterium cake (diameter 5mm) from the fungal pathogen colony edge of activation 3 days, it is placed in PDA In the center of the plate, scrape the lawn of Bacillus Velez M173 activated for 1 day, inoculate it in a parallel line at 2 cm on both sides of the pathogenic bacteria cake, and incubate at a constant temperature of 28 °C.
  • the formula for calculating the antibacterial rate is as follows:
  • Bacterial inhibition zone width (inhibition zone diameter-Beles bacillus thalline diameter)/2;
  • Bacterial antagonism index width of inhibition zone / cell radius of Bacillus velei.
  • Bacillus Velez M173 has a better inhibitory effect on fungal pathogens and bacterial pathogens.
  • the bacterial strain Bacillus Velez M173 of the present invention has good application prospects in the antibacterial aspects of crops, especially in the aspects of resistance to botrytis cinerea, fusarium wilt, damp blight, and bacterial blight.
  • Embodiment 20 Veles bacillus peanut seed coating agent
  • Conventional seed coating preparation Conventional seed coating formula: 5% sucrose, 10% PVPK30, 3% PEG, 2% dispersant MF, 5% polyethylene glycol. Each component is proportioned according to the mass fraction. A seed coating agent of 1 billion CFU/mL was prepared, and peanut seeds of similar size and texture were added to the microbial seed coating agent at an amount of 1:100 (microbial seed coating agent to seed weight ratio), and shaken to make the strain evenly adsorbed on the seeds. The effect of M173-coated peanuts on its germination and seedling growth was tested.
  • Table 34 The number of bacteria on the surface of coated seeds of each treatment
  • M173 had the best flower growth vigor after coating, and the plant height, shoot dry weight, and underground dry weight all performed the best, but there was no significant difference with CK.
  • Bacteria sample M173.
  • Chemical drug control fludiox and Jingjia cream suspension seed coating agent.
  • Conventional seed coating preparation Conventional seed coating formula: 5% sucrose, 10% PVPK30, 3% PEG, 2% dispersant MF, 5% polyethylene glycol. Each component is proportioned according to the mass fraction. A seed coating agent of 1 billion CFU/mL was prepared, and corn seeds of similar size and texture were added to the microbial seed coating agent at an amount of 1:100 (weight ratio of microbial seed coating agent to seed), and shaken to make the strain evenly adsorbed on the seeds. The effect of M173 coated corn on its germination and seedling growth was tested.
  • the auxiliary agent control is a blank seed coating agent in which the microorganisms in the seed coating agent are partially filled with maltodextrin; the chemical drug is fludioxon ⁇ Jingjia cream, diluted 10 times, and coated at 1:80.
  • the seeds after seed dressing were dried and planted in sterilized soil, 3 trees/pot, 6 pots/repeat, 3 repeats/treatment.
  • the corn after planting is watered pathogenic bacteria 10 ⁇ 6cfu/ml, 50ml/ pot. Add water every day to keep the soil humidity above 90%.
  • Water and fertilizer management Water 1 ⁇ NPK water-soluble fertilizer on the seventh day.
  • Grade 0 The whole plant grows normally and has no disease.
  • Grade 1 The aboveground and underground parts are basically normal, a small amount of lesions can be seen on the roots, and the area of lesions accounts for less than 1/4 of the root surface area, and the color of the root group is white to brown.
  • Grade 2 The aboveground and underground growth is obviously hindered, the leaf color turns yellow, the plant height is only 3/4 of that of the control, the lateral roots are few and short, and there are no fibrous roots.
  • Grade 3 The growth of aboveground and underground parts is extremely abnormal, the aboveground part can be yellow and dry, the plant height is only 1/2 of the control, the lateral roots are extremely small, the area of diseased spots accounts for 1/2-3/4 of the total root area, and the color of the root group is brown with white.
  • Grade 4 Germination, but no seedlings, almost suffocated, the area of disease spots accounts for more than 3/4 of the total root surface area, and the root is brown.
  • Disease index (%) ( ⁇ (number of plants in each disease grade ⁇ number of grades)/(total number of plants ⁇ number of highest grades)) ⁇ 100%.
  • Relative control effect (%) ((the disease index of the negative control group-the disease index of the experimental group)/the disease index of the negative control group) ⁇ 100%.
  • Tomato varieties Xinxing 101, Guangdong Kenong Biotechnology
  • Muen product M173 bacterial agent (the number of bacteria is 10 billion cfu/mL); competing product: Zhuorun.
  • Ralstonia solanacearum R.s (Ralstonia solanacearum)
  • the pathogenic strain is a highly pathogenic strain isolated from the diseased tomato plants in the Huanong experimental field, and its number is MN R.S2.
  • Ralstia solanacearum R.s (Ralstonia solanacearum) was activated from a glycerol tube onto a TTC plate (recipe: glucose 5g, peptone 10g, hydrolyzed complex protein 1g, agar 15g, add water to 1L, sterilize at 121°C for 20min, cool to 60°C and add TTC to make the final concentration 0.005% (W/V)).
  • the formula is: 20g sucrose, 5g bacteriological peptone, 0.5g K2HPO4, 0.25g MgSO4, add water to 1L, adjust to pH 7.0-7.2, sterilize at 121°C for 20min), cultivate on a shaker for 8-16h, and then dilute R.s to 3*10 according to the absorbance value of OD600 6 CFU/mL, the R.s bacteria suspension was prepared for use.
  • the fermentation process of the M173 strain was optimized, the optimal ratio of carbon and nitrogen sources was selected and the pH environment was controlled, so that the M173 could be fermented to a bacterial count of 10 billion cfu/mL.
  • the optimized medium is: 30 ⁇ 50g/L carbon source (starch hydrolysis solution, corn flour, glucose or sucrose selection one), nitrogen source 40 ⁇ 60g/L (selected one of the soybean meal or protein ⁇ ), and inorganic salt (including 2 ⁇ 5g/L and magnesium sulfate 0.5 ⁇ 2g/L, and sodium carbonate 1 to 2 to 2 to 2 to 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2. g/L), trace element 0.01-0.02g/L (Znso4 and/or mnso4);
  • the fermentation process is as follows: taking a 50L fermenter as an example, the pH is adjusted to 7 ⁇ 0.2 with 2M dilute sulfuric acid at the beginning of the fermentation, and the pH is controlled between 6-8 during the fermentation process; the dissolved oxygen is increased in the early stage of fermentation, the air volume is 0.5-1.2vvm, and the speed is 500-600rpm; the dissolved oxygen is controlled in the later stage of fermentation, the air volume is 0.2-0.5vvm, and the speed is 200-300rpm.
  • the 16d tomato seedlings were gently shaken off the soil on the root surface, and then planted in non-sterilized soil, 1 plant per pot, 10 pots were 1 treatment, and repeated 3 times.
  • Set negative control group, positive control group and bacterial agent treatment group according to different treatment solutions.
  • the treatment solution of the negative control group is clear water
  • the treatment solution of the positive control group is a 1000-fold dilution
  • the treatment solution of the bacterial agent treatment group is the biocontrol bacterial agent prepared in 4.2.
  • each seedling of each treatment group was poured into 50 mL of R.s bacterial suspension prepared by 4.2 (the positive control group added an appropriate amount of bactericidal granules to the bacterial suspension, so that the final concentration of watering was 1000 times), and the tomato seedlings were moved to the 32 ° C greenhouse disease shed (not airtight) to promote disease.
  • Plant disease is divided into the following 5 grades:
  • the control effect of M173 on tomato bacterial wilt increased with the increase of dosage, the control effect of 1L/mu on tomato bacterial wilt was 53.64%, and the control effect of 2L/mu was 77.27%. M173 has a better growth promoting effect, and the dry weight growth rate of 2L/mu is 57.72%.
  • the control effect of the competing product Zhuorun is relatively average, at 12.73%, and the control effect of chemical drugs is 41.72%.
  • M173 prevents dead bitter gourd trees and promotes growth
  • This example provides a case where Bacillus Velez M173 is used in a bitter melon test field.
  • test site was selected in a large area of bitter gourd close to the field. In previous years, there were many dead trees in the planting of tomatoes. The test site was selected to be 3 ridges, and 1 ridge was set up with 1 replicate. The soil and fertilizer conditions, light, irrigation, and terrain in 1 replicate were consistent.
  • the method of using M173 in the test field is as follows:
  • the plant height was investigated 14 days after the first treatment (Figure 20).
  • the plant heights of M173, Zorun, and Metalapex ⁇ Hymexaz all increased compared with CK.
  • the effects of M173 200 times and Zorun 400 times were better than other treatments, and the growth-promoting effect of Metalaxe ⁇ Hymexazol was 400 times lower than that of M173.
  • M173 has obvious growth-promoting effect on bitter melon, and the growth-promoting effect of 200 times is better than 400 times. At the same dose, the growth-promoting effect of M173 on balsam pear was better than that of Zhuorun, especially in the number of fruit hanging.
  • the growth-promoting effect of low dose of M173 on balsam pear was equivalent to that of mefen-hymexazol, but M173 was significantly more than that of melon-hymexazol in the number of fruit-hanging balsam pear.
  • M173 prevents dead trees of Luo Han Guo
  • Luo Han Guo is very prone to dead trees at the slow seedling stage.
  • This example provides a case of using Bacillus Velez M173 in a Luo Han Guo experimental field.
  • the basic situation of the experimental field the planting area of Luo Han Guo in the experimental field is 200 mu. Before transplanting, winter melons were planted in the previous stubble, and Luo Han Guo is very prone to dead trees during the slow seedling stage after transplanting. How to make Luo Han Guo go through the slow seedling stage is a pain point for farmers.
  • the method of using M173 in the test field is as follows:
  • Proportion of normal plants number of normal plants/total number of plants*100
  • M173-1 ml/plant can increase the proportion of normal plants of Luo Han Guo by 23%, and increase the survival rate of 41%
  • M173-2 ml/plant can increase the proportion of normal plants of Luo Han Guo by 20%, and increase the survival rate of 39%
  • Example 24 Field experiment on bacterial wilt (capsicum bacterial wilt & tomato bacterial wilt)
  • This example provides a case where Bacillus Velez M173 is used in a pepper test field.
  • the method of using M173 in the test field is as follows:
  • Plant height survey randomly select 10 plants to investigate plant height
  • fruit set number survey count the total number of fruit set in the plot. Each plot has 40 peppers.
  • Plant height growth rates are: 173-2 liters/mu 17.5%, 173-1 liters/mu 7.4%; Zhuorun 11.4%
  • This example provides a case where Bacillus Velez M173 is used in a tomato solanacear test field.
  • the method of using M173 in the test field is as follows:
  • Control effect (CK incidence rate - treatment incidence rate) / CK incidence rate ⁇ 100%
  • Embodiment 25.M173 prevents orchid stem rot
  • This example provides a case of the use of Bacillus Velez M173 in orchids.
  • the address of the experimental field Shigou Town, Sihui City, Zhaoqing City; orchid variety: Molan; basic conditions of the experimental field: the pathogenic bacteria of the stem rot of the orchid in the experimental field is Fusarium oxysporum, and the seedling tray carries the pathogen.
  • the method of using M173 in the test field is as follows:
  • the second incidence survey shows ( Figure 28, Figure 29), the control effect of M173 200-fold dilution and 400-fold dilution on orchid stem rot is basically the same, there is no dose difference, and the control effect is more than 40%.
  • Fig. 30, Fig. 31, Fig. 32 and Fig. 33 respectively show the growth of orchids treated in each group (M173 (400-fold dilution), M173 (200-fold dilution), Zhuorun, 3% metalafrost ⁇ hymexazol).
  • the control effect of M173 reached more than 50% after the third spraying, and the drug effect decreased to more than 40% after the fourth spraying.
  • the M173 Bacillus Veles of the present invention has a better biological control effect on orchid stem rot.

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Abstract

提供一种贝莱斯芽孢杆菌以及包含其的组合物，植物促生剂，生物培养物和用于植物的生长基质。还涉及它们的用途，具体来说，贝莱斯芽孢杆菌M173制备成种衣剂后，能够促进植物生长发芽，并显著提高其对病原菌的防效。并且，将其施用于作物，能够缓解作物在苗期的死棵情况。特别的，无论盆栽实验还是大田实验，都证实了本发明的贝莱斯芽孢杆菌M173对真菌，细菌病害具有显著的防效，尤其是青枯病和茎腐病的防治。因此，本发明的贝莱斯芽孢杆菌M173具有较为广泛的应用前景。

Description

一种贝莱斯芽孢杆菌及其应用
相关申请的交叉引用
本申请要求申请日为2022年1月21日的中国专利申请(申请号为202210082910.3)的优先权，该专利申请的全部内容在此被援引加入本文。
技术领域
本发明涉及微生物及病害防治技术领域，具体而言，涉及一种贝莱斯芽孢杆菌及其应用。
背景技术
在农业生产中，化肥和农药的大量施用造成了土壤退化、农作物质量与产量下降、生态环境恶化等问题，威胁我国食品及环境安全。因此利用微生物的生理性质，提供生产作物生长所需要的养分，或者提高土壤的颗粒性，或者抵抗环境中的病虫害，在安全优质农产品生产和生态环境保护中的地位不断提高。
多数农作物在种子萌发和早期幼苗阶段对环境胁迫最为敏感。刚发芽的植物根接触肥料的速度，对于植物摄取肥料中存在的营养物的能力以及植物在前三周的初始发育和生长的成功性具有重要意义。植物苗期移栽容易出现伤口，导致病原菌容易侵染，使得移栽的作物存活率较低。
土壤微生物产生的生物膜，有助于提升植物根际竞争力及根位定殖优势，有报道，微生物通过产生铁载体与植物病原体竞争，使得植物生物体可直接利用的铁离子，从而间接保护植物免受病原体的侵害。微生物可以通过诱导植株活性氧、抗氧化剂和植物激素的积累，减轻病原体对植株的负面影响，诱导植物提高植物相关防御基因的表达和某些防御相关酶来增强植物对病原体的基础免疫。微生物可以通过其鞭毛蛋白、脂肽类物质、脂多糖、铁载体等均能够作为生物激发子诱导植物产生系统抗性。
微生物菌剂施用于土壤时，作用于作物根系时，需要考虑土壤环境是否适合微生物的定殖，如酸碱性、干旱、盐碱性；此外，微生物菌剂较难应用的部位为作物地上部，即叶表、果实等部位，由于叶面相对于根系环境在应用时要要考虑叶境下高温、干旱并具紫外线等极端环境，应用于叶喷、喷雾施用方式也需考虑 微生物抗高温、抗旱、抗氧化并在作物叶表中定殖以发挥生物作用。
生物制剂产品的物理形式也是必须考虑的非常重要的因素。微生物要形成产品，不仅需要具备长期保持存活的能力，而且还必须具备能够在环境中生存并发挥其预期功能的能力。因此，具有上述特性的菌株将具有重要的应用前景。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
本发明的微生物产品可以促进植物苗期移栽的存活率，并且和腐殖酸产品配置使用可以显著促进植物的生长，有效防治植物病原菌病害和真菌性病害；通过实验也证实本发明的微生物产品适用于盐碱地，改良土壤环境；具有抗旱、耐寒的性能；此外还可以有效缓解药害；本发明的微生物产品已经验证至少12个月的时间内保持了细胞活力，而没有重大损失。在盆栽和大田试验中的应用中，它已显示出对治疗不同植物致病真菌引起的疾病以及刺激植物生长的功效。在田间试验中，它也显示出了治疗植物病原细菌，特别是西红柿青枯的功效。
在第一方面，本发明提供了一种贝莱斯芽孢杆菌M173(Bacillus velezensis)或其变体或其后代，所述贝莱斯芽孢杆菌M173保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61434。
在某些实施方案中，所述变体或后代与所述贝莱斯芽孢杆菌M173具有相似或相同的功能。
在某些实施方案中，所述贝莱斯芽孢杆菌M173的变体或后代与所述贝莱斯芽孢杆菌M173具有相似或相同的生理生化特性。
在某些实施方案中，所述贝莱斯芽孢杆菌M173具有选自下列的一项或多项功能：
(1)能够提高植物苗期(例如，苗期移栽)存活率；
(2)能够促进植物各个阶段(例如，幼苗期，缓苗期，生长期)的生长(例如，增加株高，茎粗)；
(3)能够增加植物结果的产量；
(4)能够促进植物器官生长(例如，根，茎，叶)；
(5)能够提高植物对逆境环境的抗性；
(6)能够提高植物对病原菌的抗性；
(7)(1)至(6)。
在另一方面，本发明提供了一种组合物，其包含如前所述的贝莱斯芽孢杆菌或其变体或其后代。
本领域技术人员可以理解，当所述贝莱斯芽孢杆菌与下述的任意一项组分在一起使用时，意味着该组合物不同于在自然界中发现的任何生物体。
在某些实施方案中，所述贝莱斯芽孢杆菌可以是固体形式，例如干燥的或冷冻干燥的培养物制剂。
在某些实施方案中，所述组合物还包含一种或多种另外的生物防治剂，一种或多种化学药物，或其任意组合。
在某些实施方案中，所述组合物还包含农业上或园艺上可接受的稀释剂，填充剂、溶剂、自发性促进剂、载体、乳化剂、防腐剂、分散剂、防冻剂、增稠剂、佐剂，或其任意组合。
在某些实施方案中，分散剂和/或乳化剂包括常规用于活性农业化学成分制剂中的所有非离子、阴离子或阳离子分散剂。可优选使用非离子或阴离子分散剂，或非离子或阴离子分散剂的混合物。特别合适的非离子分散剂为环氧乙烷-环氧丙烷嵌段聚合物、烷基酚聚乙二醇醚和三苯乙烯基酚聚乙二醇醚，及其磷酸化或硫酸化衍生物。特别合适的阴离子分散剂为木素磺酸盐、聚丙烯酸盐和芳基磺酸盐-甲醛缩合物。
在某些实施方案中，防腐剂的实例包括但不限于双氯酚和苄醇半缩甲醛。
在某些实施方案中，农业上或园艺上可接受的稀释剂的实例包括单糖、多糖、糖蜜、树胶、木素磺酸盐、甘油、山梨醇、丙二醇的水溶液，水，植物油和矿物油；载体可包括诸如藻酸珠、硬质小麦粉(淀粉)颗粒、硅石、黏土、黏土矿物、明胶、纤维素、纤维素衍生物、亚氯酸钙和滑石粉的固体。在一些实施方案中，载体可是多孔固体，例如硅藻土、活性碳、(例如动物骨碳)、泥炭、蛭石、褐煤、木屑和玉米穗轴。
在某些实施方案中，本发明内的组合物可被制备为：水悬浮液；稳定的液体悬浮液；乳油(emulsifiable concentrate)；胶囊；可溶性或可湿性粉末；水性悬浮剂(aqueous  flowable)；干悬浮剂(dry flowable)；可湿性颗粒；可湿性分散颗粒；以及本领域技术人员所知的类似物。
在某些实施方案中，所述另外的生物防治剂选自：细菌，真菌(例如，酵母)，病毒，昆虫，线虫，或其任何组合。
在某些实施方案中，所述细菌选自芽孢杆菌属，乳酸杆菌属，双歧杆菌属，丙酸杆菌属，链球菌属，乳球菌属，片球菌属，肠球菌属，葡萄球菌属，或其任何组合。
在某些实施方案中，所述芽孢杆菌属的细菌选自：贝莱斯芽孢杆菌(Bacillus velezensis)，枯草芽孢杆菌(Bacillus subtilis)，短小芽孢杆菌(Bacillus pumilus)，凝结芽孢杆菌(Bacillus coagulans)，或其任何组合。
在某些实施方案中，所述另外的生物防治剂是保藏编号为GDMCC No.61962的短小芽孢杆菌(Bacillus pumilus)，所述短小芽孢杆菌保藏于广东省微生物菌种保藏中心。
在某些实施方案中，所述组合物中所述贝莱斯芽孢杆菌和所述短小芽孢杆菌的混合体积比例为1:5至5：1(例如，1：5；1：4；1：3；1：2；1：1；2：1；3：1；4：1；5：1；)。
在某些实施方案中，所述组合物中贝莱斯芽孢杆菌和短小芽孢杆菌的混合体积比例为2:1。
在某些实施方案中，所述酵母选自酿酒酵母(Saccharomyces cerevisiae)，布拉氏酵母(Saccharomyces boulardii)，马克斯克鲁维酵母(Kluyveromyces marxianus)，或其任何组合。
在某些实施方案中，所述化学药物选自：除草剂，杀虫剂，抗菌剂(例如，真菌，细菌)，抗病毒剂，植物生长调节剂，抗生素，肥料，或其任意组合。
在某些实施方案中，所述抗菌剂选自霜霉威盐酸盐、多菌灵、氟啶胺、百菌清、五氯硝基苯、福美双、三乙膦酸铝、精甲霜灵、精甲霜灵咯菌腈，或其任意组合。
在某些实施方案中，所述杀虫剂选自噻虫嗪、呋虫胺、精高效氯氟氰菊酯、丙溴辛硫磷，或其任意组合。
在某些实施方案中，所述肥料选自腐殖酸类肥料(例如，根罗)尿素、硫酸铵、 欢之田、安糖可力、磷酸二氢钾，或其任意组合。
在某些实施方案中，所述组合物为固体形式，液体形式，粉末形式，或其任意组合。
在另一方面，本发明提供了一种生物培养物，其包含如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物。
在某些实施方案中，所述生物培养物还包含固体或液体培养基，或所述培养基的组分。
在某些实施方案中，所述生物培养物还包含贝莱斯芽孢杆菌M173的活细胞。在某些实施方案中，所述活细胞以孢子形式存在。在某些实施方案中，所述孢子存在于贝莱斯芽孢杆菌M173的液体培养基中。
在某些实施方案中，所述生物培养物包含贝莱斯芽孢杆菌M173的细胞和培养物的上清液。
在某些实施方案中，所述培养基包含碳源(例如，淀粉水解液、玉米粉、葡萄糖或蔗糖)，氮源(例如，豆粕，蛋白胨)，无机盐(例如，磷酸氢二钠，硫酸镁，碳酸钠)，微量元素(例如，ZnSO4，MnSO4)，或其任意组合。
在某些实施方案中，所述培养物还包含贝莱斯芽孢杆菌M173的衍生物或其子代培养物。
在某些实施方案中，所述衍生物或子代培养物选自代谢产物(例如，环二肽，环三肽，芽孢杆菌霉素，表面活性素)，激素(例如，IP，GA3，IPA，IAA，Me-SA，SA，IBA)，酶，细胞结构成分，或其任何组合。
在某些实施方案中，所述培养物还包含提供营养的成分。
在某些实施方案中，所述提供营养的成分选自蛋白质，碳水化合物，脂肪，益生菌，酶，维生素，免疫调节剂，乳汁替代物，矿物质，氨基酸，或其任何组合。
在另一方面，本发明提供了一种植物促生剂，其包含如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物。
在某些实施方案中，所述植物促生剂还包含一种或多种另外的生物防治剂，一种或多种肥料，或其任意组合。
在某些实施方案中，所述肥料选自腐殖酸类肥料(例如，根罗)或氨基酸水溶肥。
在另一方面，本发明提供了一种用于防治植物病原性细菌和/或促进植物生长的生物防治剂，其包含如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物。
在另一方面，本发明提供了一种用于植物的生长基质，其包含如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂；
在某些实施方案中，所述生长基质还包含沙子，土壤，惰性颗粒基质(例如，蛭石)；或其任意组合。
在另一方面，本发明提供了一种农药组合物，其包含如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂。
在某些实施方案中，所述农药组合物还包括一种或多种生物防治剂和/或一种或多种化学药物(例如，农药)。
在另一方面，本发明提供了一种经如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂，或如前所述的农药组合物处理过的植物、植物组织或植物器官。
在某些实施方案中，所述处理包含浸根、叶喷、喷雾、堆肥、浸种、包衣、大田漫灌、滴灌植物或植物器官，涂抹植物或植物器官，滴加植物或植物器官。
在某些实施方案中，所述植物器官包含根、茎、叶、花、果实和种子。
在某些实施方案中，所述植物组织包含分生组织，保护组织，基本组织，和输导组织。
在某些实施方案中，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科。
在某些实施方案中，所述植物具有选自下列的一项或多项特征：
(1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
(2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
(3)所述豆科植物选自大豆，花生，或其任意组合；
(4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
(5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
(6)所述兰科植物选自兰花。
在某些实施方案中，所述处理包括灌根，浸种，种子包衣。
在另一方面，本发明提供了一种提高植物苗期(例如，缓苗期移栽)存活率的方法，所述方法包括：将如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂或如前所述的生物防治剂或如前所述的农药组合物处理所述植物。
在某些实施方案中，所述处理选自灌根，浸根、叶喷、喷雾、堆肥、浸种、种子包衣、大田漫灌、滴灌。
在某些实施方案中，所述贝莱斯芽孢杆菌M173是通过选自下列的一项或多项特征来提高植物苗期存活率：
(1)提高植物苗期移栽存活率；
(2)促进植物生长(例如，增加株高，茎粗)；
(3)增加植物结果的产量；
(4)促进植物器官生长(例如，根，茎，叶)；
(5)提高植物对逆境环境的抗性；
(6)提高植物对病原菌的抗性；
(7)(1)至(6)。
在某些实施方案中，所述逆境条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合。
在某些实施方案中，所述病原菌选自病原细菌，病原真菌或病毒。
在某些实施方案中，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌 (Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合。
在某些实施方案中，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)。
在另一方面，本发明提供了一种将如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂，或如前所述的农药组合物在预防和/或治疗由病原菌引起的植物病害中的用途，或者在预防和/或缓解由非病原菌条件引起的植物损伤或坏死中的用途。
在某些实施方案中，所述病原菌选自病原细菌，病原真菌或病毒。
在某些实施方案中，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌 (Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合。
在某些实施方案中，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)。
在某些实施方案中，所述非病原菌条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤、碱性土壤、高温环境、土壤肥力不足，或其任意组合。
在某些实施方案中，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科。
在某些实施方案中，所述植物具有选自下列的一项或多项特征：
(1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
(2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
(3)所述豆科植物选自大豆，花生，或其任意组合；
(4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果， 或其任意组合；
(5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
(6)所述兰科植物选自兰花。
在另一方面，本发明提供了一种如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂，或如前所述的农药组合物在提高植物对病原菌或逆境条件的抗性中的用途，或者在促进植物生长中的用途。
在某些实施方案中，所述病原菌选自病原细菌，病原真菌或病毒。
在某些实施方案中，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合。
在某些实施方案中，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软 腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)。
在某些实施方案中，所述逆境条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤、碱性土壤、高温环境、土壤肥力不足，或其任意组合。
在某些实施方案中，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科。
在某些实施方案中，所述植物具有选自下列的一项或多项特征：
(1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
(2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
(3)所述豆科植物选自大豆，花生，或其任意组合；
(4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
(5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
(6)所述兰科植物选自兰花。
在另一方面，本发明提供了一种预防和/或治疗由病原菌引起的植物病害，或者预防和/或缓解由非病原菌条件引起的植物损伤或坏死的方法，所述方法包括：将如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂，或如前所述的农药组合物施用于植物、植物组织或植物器官。
所述的贝莱斯芽孢杆菌和其组合物的施用方法是普通的技术人员所熟悉的。这些方法的对象可以是种子、幼苗、植物、作物、植物部分、花、果实、植物营养部分(例如种块(seed tuber)和植物扦插(plant cutting))、用于培养植物材料的土壤和人工基质系统。
在某些实施方案中，所述施用包含浸根、叶喷、喷雾、堆肥、浸种、包衣、大田漫灌、滴灌植物或植物器官，涂抹植物或植物器官，滴加植物或植物器官。
在某些实施方案中，所述植物器官包含根、茎、叶、花、果实和种子。
在某些实施方案中，所述植物组织包含分生组织，保护组织，基本组织，和输 导组织。
在某些实施方案中，所述病原菌选自病原细菌，病原真菌或病毒。
在某些实施方案中，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合。
在某些实施方案中，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)。
在某些实施方案中，所述非病原菌条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合。
在某些实施方案中，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科。
在某些实施方案中，所述植物具有选自下列的一项或多项特征：
(1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
(2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
(3)所述豆科植物选自大豆，花生，或其任意组合；
(4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
(5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
(6)所述兰科植物选自兰花。
在另一方面，本发明提供了一种提高植物对病原菌或逆境条件的抗性，或者促进植物生长的方法，所述方法包括：将将如前所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者如前所述的组合物，或者如前所述的生物培养物，或如前所述的植物促生剂，或如前所述的生物防治剂，或如前所述的农药组合物施用于植物、植物组织或植物器官。
所述的贝莱斯芽孢杆菌和其组合物的施用方法是普通的技术人员所熟悉的。这些方法的对象可以是种子、幼苗、植物、作物、植物部分、花、果实、植物营养部分(例如种块(seed tuber)和植物扦插(plant cutting))、用于培养植物材料的土壤和人工基质系统。
在某些实施方案中，所述施用包含浸根、叶喷、喷雾、堆肥、浸种、包衣、大田漫灌、滴灌植物或植物器官，涂抹植物或植物器官，滴加植物或植物器官。
在某些实施方案中，所述植物器官包含根、茎、叶、花、果实和种子。
在某些实施方案中，所述植物组织包含分生组织，保护组织，基本组织，和输导组织。
在某些实施方案中，所述病原菌选自病原细菌，病原真菌或病毒。
在某些实施方案中，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌 (Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合。
在某些实施方案中，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)。
在某些实施方案中，所述逆境条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合。
在某些实施方案中，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科。
在某些实施方案中，所述植物具有选自下列的一项或多项特征：
(1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
(2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
(3)所述豆科植物选自大豆，花生，或其任意组合；
(4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
(5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
(6)所述兰科植物选自兰花。
在另一方面，本申请提供了一种抑制植物的青枯病害的方法，该方法包括：将如前所述的贝莱斯芽孢杆菌M173或其变体或其后代配制成生物农药制剂；将有效量的生物农药制剂施用于被青枯病原菌感染或有感染青枯病原菌风险的植物或其部分；在某些实施方案中，所述植物是番茄或辣椒。在某些实施方案中，述生物农药制剂通过喷雾、叶喷、浸根、灌根的方式施用。
如本文中所使用的，术语“生物防治”是指通过另外的生物体来防治病原体的一种手段。
如本文中所使用的，术语，“生物防治剂”或“生防治剂”是指能够实现生物防治的生物体(例如，微生物)。在本文中，生物防治剂不仅包括对病原菌具有抗性的微生物，还包括对植物健康、生长、活力、胁迫反应或产量具有有益效果的微生物。生物防治剂的施用途径包括喷洒施用、土壤施用(例如，灌根)和种子处理(例如，种子包衣)。
本发明的目的在于提供一种菌剂组合物及其应用，且本发明提供的贝莱斯芽孢杆菌为可商品化的生防产品，易于培养、适于工业化生产。
本发明是这样实现的：
本发明提供了一种菌剂组合物在防治如下病害中的应用，病害选自如下病害中的至少一种：植物细菌病害、植物真菌病害、植物土传病害和植物卵菌病害中的应用；
菌剂组合物包括：贝莱斯芽孢杆菌和短小芽孢杆菌M101；
贝莱斯芽孢杆菌物种的细菌菌株M173于2021年1月15日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC：No.61434；
短小芽孢杆菌M101于2021年10月22日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC：No.61962。
本发明提供的贝莱斯芽孢杆菌分离自云南省玉溪市(北纬39°54′39.33″，东经116°24′48.18″)的烟草种植地采集农业土壤。于2021年1月15日保藏于广东 省微生物菌种保藏中心，保藏编号为：GDMCC No.61434，保藏地址：广州市先烈中路100号大院59号楼五楼。
分离的菌株是一种产芽孢的革兰氏阳性细菌，将其接种于Luria-Bertani培养基中，30℃培养1d。其菌落多呈白色，表面和边缘粗糙，有粘性。对该菌株的序列进行16S序列片段测定(扩增引物与测序引物均为27F：5'-AGAGTTTGATCCTGGCTCAG-3'与1492R：5'-GGTTACCTTGTTACGACTT-3')，测定结果参照SEQ ID NO.1所示，通过BLAST同源比对，确定该菌株为贝莱斯芽孢杆菌(Bacillus velezensis)，命名为贝莱斯芽孢杆菌M173。
SEQ ID NO.1：

短小芽孢杆菌M101菌株的样本采集自广东省广州市的水中。已送至广东省微生物菌种保藏中心(GDMCC)进行保藏，保藏地址：广州市先烈中路100号大院59号楼五楼，保藏日期：2021年10月22日，保藏编号：GDMCC：No.61962。
分离的菌株将其涂布于R2A固体培养基上，于30℃恒温培养箱中培养，1天后挑取单菌落，继续接种于R2A固体培养基上，于30℃恒温培养箱中倒置培养1天，然后观察菌落形态，并用光学显微镜观察菌体形态(1000×)。
该菌株为革兰氏阳性菌，菌落呈不透明的乳白色，形状为圆形。菌株呈杆状，圆末端，单个或呈短链排列，长约2.0微米。能运动，芽孢1.0～1.2×1.5～2.0微米，椭圆形。
随后，对该菌株的序列进行了16S序列片段(扩增引物与测序引物为27F：5'-AGAGTTTGATCCTGGCTCAG-3'与1492R：5'-GGTTACCTTGTTACGACTT-3')测定，测定结果见序列表SEQ ID NO.2，16SrRNA序列同源性分析、系统发育分析，通过BLAST同源比对，我们确定该菌株最近的种属为短小芽孢杆菌(Bacillus pumilus)，并命名为短小芽孢杆菌M101。
SEQ ID NO.2：

本发明提供的贝莱斯芽孢杆菌可以高效、稳定地实现植物细菌病害、植物真菌病害、植物土传病害和/或植物卵菌病害防治，其形成生物被膜能力强，易定植， 适于工业化生产，商品化前景巨大。此外，本发明提供的贝莱斯芽孢杆菌能提升种子在存有大量病原菌的土壤环境中的发芽率，具有良好的促生作用。
在本发明应用较佳的实施方式中，上述植物土传病害包括不限于尖镰孢霉冬瓜专化型(Fusarium oxysporum f.sp.cucurmerimum)、青枯劳尔氏菌、立枯丝核菌、镰刀菌、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、番茄灰霉病菌(Botrytis cirerea)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)和/或烟草疫霉病菌(Phytophthoranicotianae)引起的植物土传病害。
在一种可选的实施方式中，镰刀菌为小麦禾谷镰刀菌。
在一种可选的实施方式中，应用温度为2-40℃；更在一种可选的实施方式中，应用温度为20-35℃。
在本发明应用较佳的实施方式中，上述植物细菌病害包括不限于青枯劳尔氏菌、(Bacillus subtilis)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)、和/或魔芋软腐病菌(Erwiniacarotovora)引起的细菌病害；
在一种可选的实施方式中，应用温度为2-40℃；更在一种可选的实施方式中，应用温度为20-35℃。
在本发明应用较佳的实施方式中，上述植物真菌病害或植物卵菌病害包括不限于立枯丝核菌、镰刀菌、油菜菌核病菌(Sclerotiniasclerotiorum)、尖镰孢霉冬瓜专化型(Fusarium oxysporum f.sp.cucurmerimum)、番茄灰霉病菌(Botrytis cirerea)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌 (Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)和/或烟草疫霉病菌(Phytophthoranicotianae)引起的植物病害。
在一种可选的实施方式中，镰刀菌为小麦禾谷镰刀菌。
在一种可选的实施方式中，应用温度为2-40℃；更在一种可选的实施方式中，应用温度为20-35℃。例如28℃或30℃。
本发明还提供了
一种菌剂组合物在促进植物生长中的应用，菌剂组合物包括：贝莱斯芽孢杆菌和短小芽孢杆菌M101；贝莱斯芽孢杆菌物种的细菌菌株于2021年1月15日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC：No.61434；
短小芽孢杆菌M101于2021年10月22日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC：No.61962。
在本发明应用较佳的实施方式中，上述植物为禾本科作物或经济作物。
在一种可选的实施方式中，禾本科作物包括不限于玉米、小麦、稻、高粱、大麦、燕麦、黑麦、谷子、黍、稗、荞麦。
经济作物选自如下经济作物中的至少一种：茄科、蔷薇科、芸香科、芭蕉科、葫芦科、蝶形花科、菊科、百合科、姜科、西番莲科、凤梨科、五加科和仙人掌科；
在一种可选的实施方式中，茄科选自如下至少一种的茄科作物：马铃薯、辣椒和番茄；蔷薇科选自如下至少一种的蔷薇科作物：草莓和木瓜；芸香科选自如下的芸香科作物：柑橘；芭蕉科选自如下的芭蕉科作物：香蕉；葫芦科选自黄瓜；蝶形花科选自大豆，菊科选自生菜，百合科选自大蒜，姜科选自生姜，西番莲科选自百香果，凤梨科选自金菠萝，五加科选自三七，仙人掌科选自火龙果。
需要说明的是，上述的作物种类仅为发明人列举的几种可选的类型，在其他实施方式，也可以根据需要进行自适应调整，并不限于上述的作物种类。
在一种可选的实施方式中，菌剂组合物中贝莱斯芽孢杆菌和短小芽孢杆菌M101的混合体积比例为1-2:1-2。菌剂组合物中贝莱斯芽孢杆菌和短小芽孢杆菌M101的混合体积比例为2:1。该配比的组合物能显著降低植株的死亡率，对植物细菌病害、植物真菌病害、植物土传病害和植物卵菌病害都有着非常好的生防潜力。
本发明还提供了一种菌剂，其包括贝莱斯芽孢杆菌和短小芽孢杆菌M101；贝莱斯芽孢杆菌物种的细菌菌株于2021年1月15日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC：No.61434；
短小芽孢杆菌M101于2021年10月22日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC：No.61962；
优选地，菌剂为贝莱斯芽孢杆菌和短小芽孢杆菌M101的发酵培养物，且发酵培养物中贝莱斯芽孢杆菌和短小芽孢杆菌M101的混合浓度为106-1010CFU/mL。例如106、107、108、109、1010CFU/mL。
本发明还提供了一种种衣菌剂，种衣菌剂包括种衣剂和上述的菌剂。种衣菌剂能提升种子在存有大量病原菌的土壤环境中的发芽率，具有良好的促生效果。
种衣剂和菌剂的混合体积比为8-10:1；优选地，菌剂中贝莱斯芽孢杆菌OD600的值为0.8-10，菌剂中短小芽孢杆菌M101OD600的值为0.8-10。
在一种可选的实施方式中，上述种衣剂配方如下：蔗糖5％，PVPK30 10％，PEG 3％，分散剂MF 2％，聚乙二醇5％。各组分按照质量分数进行配比。
制备方法如下：取50mL离心管，先按上述比例加入PVPK30和少量水，振荡溶解，再按比例加入其它试剂，溶解完全，定容，即得所需种衣剂。
本发明还提供了一种灌根剂或浸种剂，灌根剂或浸种剂包括上述的菌剂。在其他实施方式中，上述灌根剂也可以是浸根剂。
在一种可选的实施方式中，浸种剂或灌根剂是指贝莱斯芽孢杆菌和短小芽孢杆菌M101的培养液，或贝莱斯芽孢杆菌和短小芽孢杆菌M101的冻干粉。
在其他实施方式中，上述浸种剂或灌根剂也可以是贝莱斯芽孢杆菌和短小芽孢杆菌M101的悬浮液、乳液、溶液。
在一种可选的实施方式中，培养液中贝莱斯芽孢杆菌的浓度为107-108CFU/mL，培养液中短小芽孢杆菌M101的浓度为107-108CFU/mL。
发明人发现利用上述浸根剂浸根处理番茄苗等植物幼苗不仅能有效防治番茄青枯病，且其防治效果甚至优于新植霉素这一传统化学农药，可用于替代/部分替代传统化学农药，市场化前景广阔。
一种菌剂在制备种衣菌剂、灌根剂或浸种剂中的应用。
与现有技术相比，本发明的有益效果是：
本发明的提供的贝莱斯芽孢杆菌和短小芽孢杆菌M101可以高效、稳定地实现植物细菌病害、植物真菌病害、植物土传病害和/或植物卵菌病害的防治，其形成的生物被膜能力强，易定植，适于工业化生产，商品化前景巨大。此外，本发明提供的贝莱斯芽孢杆菌和短小芽孢杆菌M101能提升种子在存有大量病原菌的土壤环境中的发芽率，具有良好的促生作用。
而采用本发明提供的贝莱斯芽孢杆菌和短小芽孢杆菌M101浸根处理植物，不仅能有效防治植物病害，且其防治效果甚至优于传统化学农药，可用于替代/部分替代传统化学农药，市场化前景广阔。
发明的有益效果
本发明的贝莱斯芽孢杆菌M173具有较为突出的菌株特性。具体来说，(1)具有较强的分泌铁载体能力；(2)具有强生物被膜形成能力；(3)活菌数稳定，货架期长；M173生长快，发酵过程不易发生菌体自溶；(4)能够产生多种氨基酸以及激素，促进植物对病原体产生抗性；(5)能够产生系列环肽类化合物，帮助植物抵抗真菌、细菌和病害侵染；(6)具有较强的耐酸碱，耐高盐能力；(7)对植物有促生效果，安全系数高；(8)能与市面上常见的杀菌剂、杀虫剂和化肥兼容；(9)能够提高作物的抗逆性(例如，抗盐，抗低温，抗旱)；(10)能够缓解化药对作物的药害；(11)与腐殖酸共同使用有协同增效的促生效果。
进一步的，本发明的贝莱斯芽孢杆菌M173制备成种衣剂后，能够促进植物生长发芽，并显著提高其对病原菌的防效。并且，将其施用于作物，能够缓解作物在苗期的死棵情况。特别的，无论盆栽实验还是大田实验，都证实了本发明的 贝莱斯芽孢杆菌M173对真菌，细菌病害具有显著的防效，尤其是青枯病和茎腐病的防治。因此，本发明的贝莱斯芽孢杆菌M173具有较为广泛的应用前景。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1为M173菌株在LB培养基上的菌落形态图。
图2为M173菌剂对青枯病的剪叶生防效果图。
图3为贝莱斯芽孢杆菌M173菌株对青枯病的灌根生防效果图。
图4为贝莱斯芽孢杆菌M173菌株对禾谷镰刀菌的生防效果图。
图5为贝莱斯芽孢杆菌M173菌株的铁载体能力结果。
图6为贝莱斯芽孢杆菌M173菌株和竞品菌株的生长曲线。
图7为贝莱斯芽胞杆菌M173与其它贝莱斯芽胞杆菌的生长曲线。
图8为不同pH对M173贝莱斯芽孢杆菌M173菌株的菌数的影响。
图9为不同NaCl含量(％)对贝莱斯芽孢杆菌M173菌株的菌数生长的影响。
图10为不同组别处理对黄瓜的促生效果。
图11为不同盐浓度下施用贝莱斯芽孢杆菌M173菌株的黄瓜苗长势情况。
图12为浇灌200mM氯化钠后施用贝莱斯芽孢杆菌M173组和UTC组(未处理组)的黄瓜情况。
图13为盐害现象下黄瓜受损情况。
图14为解药害各处理的黄瓜苗长势情况。
图15为抗寒试验各处理的黄瓜苗长势情况。
图16为抗旱试验各处理的黄瓜苗长势情况。
图17为贝莱斯芽孢杆菌M173的平板抑菌效果。
图18为贝莱斯芽孢杆菌M173包衣后的花生苗的长势。
图19为贝莱斯芽孢杆菌M173包衣后的花生种子的情况。
图20为不同组别处理后苦瓜的株高。
图21为不同组别处理后苦瓜的长势，挂果数。
图22为不同组别处理后罗汉果的防死棵结果。
图23为不同组别处理试验地1后对番茄青枯防效的情况。
图24为不同组别处理试验地3后对番茄青枯防效发病率的结果。
图25为不同组别处理试验地4后对番茄青枯防效的情况。
图26为第一次发病调查中不同组别处理后兰花茎腐病的发病率情况。
图27为第一次发病调查中不同组别处理后兰花茎腐病的相对防效。
图28为第二次发病调查中不同组别处理后兰花茎腐病的发病率情况。
图29为第二次发病调查中不同组别处理后兰花茎腐病的相对防效。
图30为M173(400倍稀释)组的兰花生长情况。
图31为M173(200倍稀释)组的兰花生长情况。
图32为卓润组的兰花生长情况。
图33为3％甲霜·噁霉灵组的兰花生长情况。
序列信息
本发明涉及的部分序列的信息提供于下面的表中。
序列的描述

具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.
本实施例提供了贝莱斯芽孢杆菌M173菌株的筛选及鉴定方法。
M173菌株的筛选方法：
贝莱斯芽孢杆菌(Bacillus velezensis)M173分离自云南省玉溪市(北纬39°54′39.33″，东经116°24′48.18″)的烟草种植地采集农业土壤。
称取5g土壤，加入45mL无菌水后用涡旋震荡仪震荡5min，再梯度稀释为10-1～10-6，取10-4、10-5和10-6三个梯度的菌液进行平板涂布，涂布于Luria-Bertani(LB)固体培养基(配方为：酵母浸粉5g、胰酪蛋白胨10g、氯化钠10g、琼脂15g、水1L、pH＝7)上，每个涂布进行三个重复，于30℃恒温培养箱中倒置培养4d，4d后挑出单菌落的菌株，划线接种于新的LB平板上，30℃条件下培养1天，获得菌株纯培养，用甘油管保存于-80℃冰箱内。
M173菌株的鉴定：
将分离的菌株接种于Luria-Bertani培养基中，30℃培养1d，形态如图1所示。经鉴定，该菌株是一种产芽孢的革兰氏阳性细菌，其菌落多呈白色，表面和边缘粗糙，有粘性。
随后对该菌株的序列进行16S序列片段测定(扩增引物与测序引物为27F：5'-AGAGTTTGATCCTGGCTCAG-3'与1492R：5'-GGTTACCTTGTTACGACTT-3')，测定结果参照SEQ ID NO:1所示，通过BLAST同源比对，确定该菌株为贝莱斯芽孢杆菌(Bacillus velezensis)，命名为贝莱斯芽孢杆菌M173。将贝莱斯芽孢杆菌M173送至广东省微生物菌种保藏中心(GDMCC)进行保藏，保藏地址：广州市先烈中路100号大院59号楼五楼，保藏日期：2021年01月15日，保藏编号：GDMCC No.61434。
实施例2.
1.短小芽孢杆菌M101菌株的分离纯化。
样本采集自广东省广州市的水中。
用移液枪吸取4mL水源样本，加入到灭过菌的36mL浓度为0.1％(v/v)的吐温水中，涡旋振荡10min，获得10-1浓度稀释液。用吐温水将稀释液梯度稀释至10-4、10-5和10-6浓度，然后涂布于R2A固体培养基上，于30℃恒温培养箱中培养，1天后挑取单菌落，继续接种于R2A固体培养基上，于30℃恒温培养箱中倒置培养1天，然后观察菌落形态，并用光学显微镜观察菌体型态(1000×)。
R2A固体培养基：称取18.1g R2A琼脂培养基干粉(广东环凯生物科技有限公司)，补水至1L，121℃高压蒸汽灭菌20分钟。
2.短小芽孢杆菌M101菌株的鉴定
该菌株为革兰氏阳性菌，菌落呈不透明的乳白色，形状为圆形。菌株呈杆状，圆末端，单个或呈短链排列，长约2.0微米。能运动，芽孢1.0～1.2×1.5～2.0微米，椭圆形。
随后，对该菌株的序列进行了16S序列片段(扩增引物与测序引物为27F：5'-AGAGTTTGATCCTGGCTCAG-3'与1492R：5'-GGTTACCTTGTTACGACTT-3')测定，测定结果见序列表SEQ NO:1，16SrRNA序列同源性分析、系统发育分析，通过BLAST同源比对，我们确定该菌株最近的种属为短小芽孢杆菌(Bacillus pumilus)，并命名为短小芽孢杆菌M101。短小芽孢杆菌M101已送至广东省微生物菌种保藏中心(GDMCC)进行保藏，保藏地址：广州市先烈中路100号大院59号楼五楼，保藏日期：2021年10月22日，保藏编号：GDMCC No.61962。
实施例3.
本实施例进行贝莱斯芽孢杆菌M173发酵培养物的制备。
将实施例1获得的M173菌株活化后，进行液体发酵，制得发酵培养物。该发酵培养方法简便易操作，适于工业化生产。
a.贝莱斯芽孢杆菌M173菌株的活化。
LB固体培养基：胰蛋白胨10g，酵母粉5g，NaCl 10g，琼脂粉15g，补水至1L，121℃高压蒸汽灭菌20分钟。用涂布法将-80℃冰箱保存的M173菌株涂于LB平板上，30℃培养1天。挑取LB平板上长出的M173单菌落接种于LB液体培养基(配方为：蛋白胨10g，酵母粉5g，氯化钠5g，葡萄糖1g，加水至1L，121℃灭菌20min)中，30℃培养1天，获得M173菌种。
b.M173菌株液体发酵。
将1mL M173菌种接种于LB液体培养基，在30℃摇床无菌培养1天，获得发酵培养物。
实施例4.
本实施例进行短小芽孢杆菌M101发酵培养物的制备。
将实施例2获得的短小芽孢杆菌M101菌株活化后，进行液体发酵，制得发酵培养物。该发酵培养方法简便易操作，适于工业化生产。
a.短小芽孢杆菌M101菌株的活化。
LB固体培养基：胰蛋白胨10g，酵母粉5g，NaCl 10g，琼脂粉15g，补水至1L，121℃高压蒸汽灭菌20分钟。用涂布法将-80℃冰箱保存的M173菌株涂于LB平板上，30℃培养1天。挑取LB平板上长出的M173单菌落接种于LB液体培养基(配方为：蛋白胨10g，酵母粉5g，氯化钠5g，葡萄糖1g，加水至1L，121℃灭菌20min)中，30℃培养1天，获得M173菌种。
b.M101菌株液体发酵。
将1mL M101菌种接种于LB液体培养基，在30℃摇床无菌培养1天，获得发酵培养物。
实施例5.
本实施例进行M173、M101及其组合物对青枯劳尔氏菌MN R.s1菌株的剪叶生防效果验证实验。
(1)R.s菌悬液制备：
将青枯劳尔氏菌MN R.s1(Ralstonia solanacearum，保存在慕恩生物菌库)从甘油管中活化到TTC平板(配方为：葡萄糖5g，蛋白胨10g，水解酪蛋白1g，琼脂15g，加水至1L，121℃灭菌20min，冷却至60℃后加TTC，使终浓度为0.005％(W/V))上，培养2d后选取活性高的青枯菌落转接至SPA液体培养基(配方为：蔗糖20g，细菌学蛋白胨5g，K2HPO4 0.5g，MgSO4 0.25g，加水至1L，调至pH为7.0～7.2，121℃灭菌20min)中，摇床上培养8-16h后再根据OD600的吸光度值将R.s稀释至107CFU/mL，制得R.s菌悬液备用。
(2)M173菌剂制备：
用分光光度计测定实施3制得的M173菌株发酵培养物在OD600处的吸光度值，并用LB液体培养基将其浓度调整至108CFU/mL，即得M173菌剂。
(3)M101菌剂制备
用分光光度计测定实施例4制得的M101菌株发酵培养物在OD600处的吸光度值，并用LB液体培养基将其浓度调整至108CFU/mL，即得M101菌剂。
(4)M173与M101菌剂制备
将上述步骤中制备的M173与M101菌剂按照体积比，分别配置成1:1、2:1、1:2三种浓度，混匀。
(5)番茄苗处理：
将11d番茄苗间苗至10株/盆后，进行剪叶处理用于初步判断M173对R.s的生防效果。按照剪叶时所用剪刀的浸泡液的不同，分别设置空白对照组、阴性对照组、阳性对照组和菌剂处理组，每组三次重复。对每株番茄苗进行剪叶前，将无菌剪刀浸泡在各组对应的浸泡液中1秒后，再用浸泡后的剪刀剪掉番茄苗子叶的一半。随后把剪叶后的番茄苗盆放置在30℃高温高湿的发病棚内培养7d。并从剪叶后第4d起，每天记录番茄苗死苗数，计算死亡率和相对防效。
各组所用的浸泡液如下：
空白对照组为清水40mL；
阴性对照组为清水和R.s菌悬液各20mL，混匀；
阳性对照组为新植霉素1000倍稀释液和R.s菌悬液各20mL，混匀；
菌剂处理组共5组，分别取步骤(3)和步骤(4)制得的菌剂20mL与R.s菌悬液20mL，混匀。
相对防效＝(阴性对照死亡率-处理组死亡率)/阴性对照死亡率×100％。
死亡率和相对防效统计结果参照表1所示，M173菌剂对青枯病的剪叶生防效果参照图2所示。结果显示，在剪叶体系下，贝莱斯芽孢杆菌M173菌剂处理组的番茄苗死亡率为18.12％，较阴性对照组番茄苗死亡率(87.78％)低，但较阳性对照组番茄苗的死亡率(16.67％)还是高。M101菌剂处理组的番茄苗死亡率为65.01％，防效略差。另外，3个菌剂混合处理组对青枯病的防治效果皆高于阳性对照，尤其是2:1组。说明M173和M101的混合使用效果优于单一使用效果，混合菌剂能显著降低番茄苗的死亡率，对番茄青枯病有着非常好的生防潜力。
表1各处理组对青枯病的剪叶生防效果
实施例6.
本实施例进行贝莱斯芽孢杆菌M173菌株、M101短小芽孢杆菌菌株以及二者的组合物对青枯劳尔氏菌R.s的灌根生防效果验证实验。本实施例使用的是青枯劳尔氏病原菌选自慕恩菌库，该病原菌编号为MN R.s1(Ralstonia solanacearum)。
按照实施例5的方法制备R.s菌悬液、M173菌剂、M101菌剂、M173与M101菌剂(体积比分别为1:1、2:1、1:2)备用。
(1)番茄苗处理
将20d番茄苗，轻轻抖去根表面泥土，将根部浸泡于处理液中10min，随即种入灭菌土中，每盆1株，6盆为1个处理，5重复。根据处理液不同设置空白对照组、阴性对照组、阳性对照组和菌剂处理组。空白对照组和阴性对照组的处理液为清水，阳性对照组的处理液为新植霉素1000倍稀释液，菌剂处理组共5组，各组对应的处理液分别为M173菌剂、M101菌剂、M173与M101菌剂(体积比分别为1:1、2:1、1:2)。将盆栽置于25-28℃环境中缓苗7d后用园艺小铲插入根部四周土中，使番茄根部受损造成伤口。然后将阴性对照组、阳性对照组和菌剂处理组的每株苗灌入50mL R.s菌悬液，空白对照组灌等量清水。
(2)观察记录
在出现症状的第一个迹象(番茄苗出现青枯症状)开始(一般为灌根后第5d开始发病)，每天观测记录一次番茄苗状态(根据分级标准记录发病情况)，至第10d-13d(视实际发病情况确定)，记录各处理每个病级的植株数，根据以下公式计算发病率和病情指数。
植株病情等级分为以下5级：
0级：无症状
1级：一片叶萎蔫
2级：2-3片叶萎蔫
3级：整株枯萎
4级：植株死亡
病情指数(％)＝(∑(每个病级的植株数×级别数)/(总植株数×最高级别数))×100％。
防治效果(％)＝((阴性对照组病情指数-实验组病情指数)/阴性对照组病情指数)×100％。
由表2和图3可知，阴性对照组的番茄苗因为仅以清水浸根而未加入任何药剂成分，其受青枯劳尔氏菌R.s感染后的发病率为41.7％，番茄苗表现出植株严重萎蔫、甚至全株枯死的现象。同阴性对照组相比，菌剂处理组的番茄苗长势更优，其中2：1组的植株发病率(12.12％)比阴性对照组降低了70.94％，对青枯病的防治效果高达65.02％，优于阳性对照(37.30％)。说明M173和M101按 照适宜比例混合后浸根处理番茄苗不仅能有效防治番茄青枯病，且其防治效果甚至优于新植霉素这一传统化学农药，可用于替代/部分替代传统化学农药，市场化前景广阔。
表2各处理组对青枯病的灌根生防效果
实施例7.
本实施例进行贝莱斯芽孢杆菌M173菌株、M101短小芽孢杆菌菌株以及二者的组合物对小麦禾谷镰刀菌的生防效果验证实验。
(1)F.g菌悬液制备：
将禾谷镰刀菌F.g(Fusarium graminearum，保存在慕恩生物菌库)在PDA平板活化，28℃培养5d后，再用CMC培养基(配方为：CMC-Na 15g，NH4NO31g，酵母膏1g，MgSO4·7H2O 0.5g，KH2PO4 1g，加水至1L，121℃灭菌20min)在25～28℃，200rpm条件下培养7d后用纱布过滤得到孢子悬液，用血球计数板计数，然后配制成浓度为106CFU/mL的孢子菌悬液。
(2)种衣剂的制备
配方：蔗糖5％，PVPK30 10％，PEG 3％，分散剂MF 2％，聚乙二醇5％。各组分按照质量分数进行配比。
取50mL离心管，先按上述比例加入PVPK30和少量水，振荡溶解，再按比例加入其它试剂，溶解完全，定容，即得所需种衣剂。
(3)种衣菌剂的制备
分别将实施例3和实施例4所得的M173菌株发酵培养物和M101菌株发酵培养物用分光光度计测量其在OD600处的吸光度，并用LB液体培养基将发酵培养物的OD600值调整为1，制得M173菌液和M101菌液。并将所得M173菌液和M101菌液按照体积比1:1、2:1、1:2制得M173与M101混合菌液备用。
分别吸取40μL上述菌液与360μL种衣剂混合，即得M173种衣菌剂、M101种衣菌剂及3组混合种衣菌剂。用等量LB液体培养基替代发酵培养物与等量种衣剂混合制得对照种衣剂。
(4)种子处理
设置空白对照组、阴性对照组和菌剂处理组。菌剂处理组用种衣菌剂对小麦种子(济麦23)进行包衣(种衣菌剂400μL/20g小麦)，使M173和/或M101附着在种子表面。空白对照组种子不做任何处理，阴性对照组种子用对照种衣剂进行包衣处理。
(5)土壤准备
将每个育苗盆内加入无菌土，其中阴性对照组和菌剂处理组的无菌土内提前拌入了20mL F.g菌悬液，空白对照组拌入等量清水。然后将包衣好的种子种入育苗盆内。
发芽后统计小麦发芽率，14d后统计小麦病情。
其病情分级情况具体为：
0级，未发病；
1级，第一叶鞘出现轻度症状(第一叶鞘出现褐色条纹至黑化，但黑化程度不超过50％)；
2级，第一叶鞘发病严重(第一叶鞘黑化程度超过50％以上)；
3级，第二叶鞘出现轻度症状(第二叶鞘出现褐色条纹至黑化，但黑化程度不超过50％)；
4级，第二叶鞘发病严重(第二叶鞘黑化程度超过50％以上)；
5级，第三叶鞘轻度发病(第三叶鞘出现褐色条纹至黑化，但黑化程度不超过50％)；
6级，第三叶鞘发病严重至植株接近死亡。
参照表3和图4，阴性对照组的小麦植株病情指数为38.89％，M173处理组的病情指数为20.01％，而组合物2:1的病情指数仅为15.43％，其相对防效为60.32％。说明M173能有效防治F.g对小麦植株的侵害，且组合物2:1较M173防效更佳。另一方面，禾谷镰刀菌F.g使得阴性对照组小麦种子发芽率降低至77.42％，而含M173菌液的处理组种子发芽率在91％以上，且组合物2:1的小麦种子发芽率高达95％以上。说明M173能有效确保植物种子在F.g环境中的发芽率，而组合物2：1能进一步提升发芽率。本发明提供的种衣菌剂的存在能有效保护小麦种子免受禾谷镰刀菌F.g的影响，促进种子发芽；且M173和M101的组合物具有进一步促进种子发芽和高效防治禾谷镰刀菌的双重功效。
表3禾谷镰刀菌的生防效果对比
实施例8.
本实施例进行贝莱斯芽孢杆菌M173菌株、M173与M101组合物生物被膜形成能力测试实验。
在确保贝莱斯芽孢杆菌M173菌株本身易培养、且能有效防治青枯劳尔氏菌和禾谷镰刀菌导致的土传病害的前提下，通过进一步验证M173、M173与M101组合物的生物被膜形成能力来检验其在田间土壤/植株叶面的定植能力，以期验证M173、M173与M101组合物的的防效稳定性和持续性。
分别将实施例3和实施例4所得的M173菌株发酵培养物和M101菌株发酵培养物用分光光度计测量其在OD600处的吸光度，并用LB液体培养基将发酵培 养物的OD600值调整为1，制得M173菌液和M101菌液。并将所得M173菌液和M101菌液按照体积比1:1、2:1、1:2制得M173与M101混合菌液备用。
分别吸取100μL上述菌液至96孔板中培养24h。用200μL PBS溶液清洗2次后将96孔板放入60℃烘箱中1h使生物膜固定，然后向孔中加入50μL 0.4％结晶紫溶液染色15min后，用200μL PBS清洗3次，最后在37℃下烘干液体，加入200μL 70％乙醇，并用酶标仪测量600nm处的吸光度，以此评价贝莱斯芽孢杆菌M173的生物被膜形成能力。整个实验过程，设置阴性对照组和菌剂处理组，其中阴性对照组往96孔板中加入LB液体培养基，菌剂处理组共5组，每组加入对应的菌液，每组设置三个平行。
表4生物被膜形成能力
根据测得的吸光度取3次重复的平均值(D)，以阴性对照组平均值的2倍作为界限值(Dc)。当菌剂处理组的平均值D处＞2×Dc时判定菌株为强生物被膜形成株；当Dc＜D处≤2×Dc时判定菌株为弱生物被膜形成株；当D处≤Dc为判定菌株为无生物被膜形成株。
由表4可知，D对＝0.24，Dc＝0.48，2×Dc＝0.96，而除M101外的其他4组菌剂处理组的平均值＞2×Dc，即M101不具备强生物被膜形成能力，而贝莱斯芽孢杆菌M173菌株是一株具有强生物被膜形成能力的菌株，且M173与M101的组合物也具备该能力。强生物被膜形成能力表征着贝莱斯芽孢杆菌M173菌株及组合物在植株土壤环境中极容易进行定植，从而更快速、稳定、持久的产生生防效果。
实施例9.
本实施例进行贝莱斯芽孢杆菌M173菌株、M101短小芽孢杆菌菌株以及二者的组合物对黄瓜立枯病的生防效果验证实验。
(1)病原菌准备
利用打孔器，在无菌环境条件下在长满立枯丝核菌培养基上打孔得到直径5mm的菌饼，将其接到PDA(配方为：马铃薯200g加水煮烂、过滤，葡萄糖20g，琼脂15g，加水至1L，121℃灭菌20min)培养基平板上，每皿一个菌饼，28℃恒温培养箱中培养7d备用。
(2)浇灌处理
黄瓜育苗7d后，向育苗盘中浇灌水溶肥，播种12d时，将其拔出用剪刀将全部须根及部分主根剪掉后，挑取长势一致的黄瓜苗用灭菌土种于小花盆中，每盆3株。将实施例3和实施例4获得的M173菌株发酵培养物和M101菌株发酵培养物用分光光度计测量其在OD600处的吸光度，并用LB液体培养基将发酵培养物的OD600值调整为0.12后，制得M173菌液和M101菌液。并将所得M173菌液和M101菌液按照体积比1:1、2:1、1:2制得M173与M101混合菌液备用。
用上述菌液浇灌黄瓜苗，每盆浇灌50mL共6×108cfu，每6盆为1个处理，每处理3重复。对照组浇灌等量清水。接菌2小时后在每盆相对位置上戳两个小孔，每孔接一个直径5mm的病原菌菌饼，然后置于28～30℃温室棚中发病7d。
将经过高温高湿发病后的黄瓜苗根部土壤清洗干净，根据病级分级标准记录发病情况，最后将清洗后的黄瓜苗放入75℃烘箱中烘干，称量干重，统计干重增长率。
病级分级标准：
0级：黄瓜苗茎基部无病斑
1级：黄瓜苗茎基部病斑所占茎围小于1/4
2级：黄瓜苗茎基部病斑所占茎围在1/4～1/2之间
3级：黄瓜苗基部病斑所占茎围大于1/2，但未破坏整个茎围
4级：黄瓜苗根茎相接处出现凹陷或断裂，腐烂变褐等症状
发病指数＝(∑(各级病株数×各病级代表数值)/(调查总株数×发病最高级数值))×100
相对防效(％)＝(对照组病级指数-处理组病级指数)/对照组病级指数×100％。
表5各处理组对黄瓜立枯病的防治效果
由表5可知，经组合物2:1发酵培养物浇灌处理的黄瓜苗病级主要介于0-1级之间，而对照组黄瓜苗病级多数处于1-2级，其黄瓜苗发病指数(44.59％)均高于处理组，组合物2:1对黄瓜立枯病的相对防效达39.78％，即本发明提供的贝莱斯芽孢杆菌M173及其与M101的组合物能有效抑制立枯丝核菌对植株的侵害。
实施例10.
本实施例进行贝莱斯芽孢杆菌M173菌株、M101短小芽孢杆菌菌株以及二者的组合物对冬瓜枯萎病的生防效果田间验证实验。
试验基地
东莞市农业科学研究中心(香蕉蔬菜基地)
实验作物：
冬瓜，品种莞研2号小冬瓜；大棚种植。
表6.试验处理

区域划分概况：
以作物棵数划分，每处理34棵苗，3重复共102颗苗(实际仍以此棚种植瓜苗数量为准)，每处理及重复组随机排列。研究所给出两方案，以实际情况为准。
实验步骤
选取相同生长时期冬瓜苗；
表7.施用时机
试验记录
(1)发病率
两次浇灌后三周回访统计，使用慕恩生物两菌剂产品,尤其是2:1，可有效降低冬瓜枯萎病病原菌尖镰孢霉冬瓜专化型(Fusarium oxysporum f.sp.cucurmerimum)的侵害，减少冬瓜枯萎病发生率，新生叶片较对照多并且长势旺盛。
表8.冬瓜枯萎病药效试验发病统计

综上所述，本发明提供的组合物不仅可以拮抗青枯劳尔氏菌、禾谷镰刀菌和立枯丝核菌，有效防治番茄青枯病、小麦赤霉病、黄瓜立枯病和冬瓜枯萎病，还能促进种子发芽、提升发芽率；并能快速定植、便于工业化生产，具有广阔的应用前景。
实施例11.M173菌株的特性测试实验
(1)贝莱斯芽孢杆菌M173铁载体能力测试
铁载体是微生物合成并分泌的一种螯合铁的用于摄取铁元素的低分子复合物，根系微生物可通过产铁载体螯合铁供给植物利用；另一方面，生防菌产铁载体可以与病原菌竞争铁元素，抑制病原微生物的生长繁殖。
1.1试验材料：
CAS培养基、MKB无铁培养基、磷酸氢二钾溶液、50ml离心管、离心机、接种环、无菌水。
1.2实验方法：
铁饥饿处理：以慕恩菌库中的一株短小芽孢杆菌(Bacillus pumilus)菌株作为阴性对照(该菌株的筛选方法以及鉴定方法参照实施例2所述的内容)，用接种环刮取供试菌株，接种到装有10mL MKB无铁培养基的50mL离心管中，30℃,200rpm振荡培养24h-72h。待菌体产生后，1000rpm离心5min，弃上清。将菌体用5mL无菌超纯水10000rpm离心5min洗涤2次，再用无菌超纯水稀释10倍获得菌悬液。
CAS平板覆盖法：取10μL菌悬液接种于无铁MKB平板，或用接种环划线接种于无铁MKB平板。每个菌株3个重复，置于30℃培养箱中培养2d。2d后，每个平板上都长出明显单菌落，待CAS培养基冷却至60℃以下，在长出菌落的平板上倒一层CAS培养基，放置1h后观察每个平板的颜色变化，24h后再观察一次，拍照记录。
1.3实验结果：
通过计算可溶性指数，即晕圈比D/d的大小，以可溶性指数为参考，分析比较生防菌分泌铁载体的能力。
表9.可溶性指数
图5和表9的实验结果显示，本发明的贝莱斯芽孢杆菌M173铁载体能力较好，优于阴性对照的菌株，阴性对照显示其不能产铁载体。
(2)贝莱斯芽孢杆菌M173生物膜
生物膜成膜性为细菌堆积后在物体表面成膜特性，具有成膜性的微生物产品菌株与田间应用定殖能力以及病害生防效果呈正相关。
制备M173菌株发酵培养物，用分光光度计测量其在OD600处的吸光度，并用LB液体培养基将发酵培养物的OD600值调整为1后，从中吸取100μL发酵培养物至96孔板中培养24h。用200μL PBS溶液清洗2次后将96孔板放入60℃烘箱中1h使生物膜固定，然后向孔中加入50μL 0.4％结晶紫溶液染色15min后，用200μL PBS清洗3次，最后在37℃下烘干液体，加入200μL 70％乙醇，并用酶标仪测量600nm处的吸光度，以此评价贝莱斯芽孢杆菌M173的生物被膜形成能力。整个实验过程，设置阴性对照组、竞品菌剂对照组和菌剂处理组，其中阴性对照组往96孔板中加入LB液体培养基，菌剂处理组加入发酵培养物(OD600＝1)，每组设置三个平行。
根据测得的吸光度取3次重复的平均值(D)，以阴性对照组平均值的2倍作为界限值(Dc)。当菌剂处理组的平均值D处＞2×Dc时判定菌株为强生物被膜形成株；当Dc＜D处≤2×Dc时判定菌株为弱生物被膜形成株；当D处≤Dc为判定菌株为无生物被膜形成株。
进一步的，根据测得的吸光度取3次重复的平均值(D)，D值越大，表明菌株成膜性越好。实验结果如表10所示。由表10可以看出，M173的吸光度平均值(D)稳定的在3.7以上，显著的高于阴性对照以及其它的对照菌株。
表10.各菌株的生物膜特性

(3)M173生长曲线
1、贝莱斯芽胞杆菌与其它细菌的生长曲线
本实施例选用的对照菌株分别为：实施例2鉴定获得的短小芽孢杆菌M101；竞品1-由市售的枯草芽孢杆菌产品分离获得的枯草芽孢杆菌、竞品2-市售的解淀粉芽孢杆菌产品分离获得的解淀粉芽孢杆菌。
带有挡板的250ml三角瓶培养基装量为50mL，培养好种子液(本发明的M173和上述三个对照菌株)按2％(v/v)接种到发酵培养基(葡萄糖5g/L，酵母粉5g/L，蛋白胨5g/L，NaCl 5g/L)，30℃，200rpm培养，定时取样检测发酵液OD600。实验结果如图6所示，结果显示，贝莱斯芽胞杆菌M173对数期最短，因此其能够快速建立功能菌群。并且，贝莱斯芽胞杆菌M173的稳定期长，因此其能够产生大量次生代谢产物。
综上，M173能够快速生长，货架期长，可分泌大量抑制病原菌的次生代谢物质，直接杀灭病菌；形成生物膜能力强，快速在作物根系形成保护层，抵御病原菌入侵，双重机制防病。
2、贝莱斯芽胞杆菌M173与其它贝莱斯芽胞杆菌的生长曲线
对于芽孢杆菌，发酵后期，当营养缺乏或条件变差时，会逐渐形成芽孢，但有相当部分芽孢杆菌会出现菌体自溶而不产芽孢的情况，对于大规模发酵形成产品有较大的挑战；本发明通过对贝莱斯芽孢杆菌M173和实施例19中记载的的贝莱斯芽孢杆菌2和贝莱斯芽孢杆菌5进行进一步的比较，综合生长曲线、产芽孢情况和自溶情况，M173综合所有因素考量，是具有开发成产品的最优菌株；
实验菌株：贝莱斯芽孢杆菌2、贝莱斯芽孢杆菌5(根据实施例1记载的筛选方法和鉴定方法获得的)；贝莱斯芽孢杆菌M173
种子培养基：LB培养基(g/L)：蛋白胨10，酵母粉5，NaCl 5；
发酵培养基：LB培养基(g/L)：蛋白胨10，酵母粉5，NaCl 5；
培养和检测方法：带有挡板的250ml三角瓶培养基装量为50mL，每个菌株备7瓶培养基，培养好的种子液按2％(v/v)接种到发酵培养基，30℃，200rpm培养基，分别于0h，4h，8h，14h，18h，22h，24h取样。
取样检测OD600、并镜检产芽孢情况。
实验结果(表11，图7)：在以LB为发酵培养基的条件下，三个菌株中，在14h前，贝莱斯芽孢杆菌2、贝莱斯芽孢杆菌5、贝莱斯芽孢杆菌M173中菌浓度(根据OD600)从高到低依次为贝莱斯芽孢杆菌2、贝莱斯芽孢杆菌5、贝莱斯芽孢杆菌M173，即14h前贝莱斯芽孢杆菌2生长最快，其次为贝莱斯芽孢杆菌5，最后为贝莱斯芽孢杆菌M173。
另外，三者的OD600均在14h取样时达到最高，但贝莱斯芽孢杆菌2、贝莱斯芽孢杆菌5在后续培养在易发生自溶，OD下降明显，而M173自溶相对较少，OD下降相对较慢，且在22h镜检时开始产芽孢。贝莱斯芽孢杆菌2、贝莱斯芽孢杆菌5在使用LB作为培养基时镜检未发现有芽孢形成。
表11：不同菌株的发酵实验结果
(4)M173相对货架期
实验方法：根据Arrhenius定律，按照公式lgC＝-(Kt/2.303)+lg C0式中，C0为t＝0时菌数，C为时间t时菌数。以lgC对t作线性回归，得到一直线，由直线斜率＝-K/2.303，由此先计算出K值，不同的温度有不同的K值。根据公式可推算出室温25℃时的K值。把室温25℃的K值，初始活菌数C0，最终在保质期内要达到的活菌数C，即可求得保藏的时间t。
将样品分别放置于35℃、45℃、55℃下储存，定期取样进行稀释涂板，计算高温模拟货架期。
根据Arrhenius定律：
lg K＝－(E/2.303RT)+lg A
K：反应速度常数；
A：频率因子；
E：为活化能；
R：气体常数(R＝8.314J/(mol*K))；
T：绝对温度
根据表12中不同温度下的菌数，按照公式可推算出自变量为温度T，并进一步计算得到K值；根据Arrhenius定律计算出25℃下的样品的衰减曲线为：lg C＝-(0.000735t/2.303)+lgC0根据衰减曲线可以计算出25℃(常温)下，贝莱斯芽孢杆菌发酵液半衰期为943.2天。
表12.不同温度下的菌数
根据加速实验的数据，计算得到在室温25℃下的K值为0.000735，实际检测出厂菌数C0为8.23×109，套入公式lg C＝－(Kt/2.303)+lg C0中，可计算得到半衰期t为943.20，即预测产品出厂2.5年后(946.20d)，菌数仍维持在4.11×109cfu/mL，满足产品保质期2年的需求。
(5)M173的绝对货架期(PH、活菌数、芽孢数、芽孢率)检测
1.活菌数测定方法
液体菌剂取10.0mL加入90mL无菌水中，静置20min，在旋转式摇床上200r/min充分振荡30min，即成母液菌悬液。
用无菌移液管分别吸取5.0mL上述母液菌悬液，加入45mL无菌水中，混匀即得10-2的稀释液。继续进行梯度稀释。每个样品取3个连续适宜的稀释度，用无菌移 液管分别吸取不同稀释度菌悬液0.1mL，加至预先制备好的固体培养基平板上，利用玻璃珠将菌悬液涂布均匀。每一稀释度重复3次，将平板置于适宜的条件下培养。
以出现20个-300个菌落数的稀释度的平板为计数标准，统计有效活菌数目。当只有一个稀释度，其平均菌落数在20个-300个之间时，则以该平均菌落数计算。若有2个稀释度，其平均菌落数均在20个-300个之间时，应按两者菌落总数之比值决定。若其比值小于等于2应计算两者的平均数；若大于2则以稀释度小的菌落平均数计算。
每克样品中活菌数＝(C/V)×M
C为某一稀释浓度下平均菌落数，V代表涂布平板时所用稀释液的体积(mL)，M代表稀释倍数。
2.芽孢数测定方法
另取1.中的母液菌悬液于80℃水浴30min，然后稀释涂板，统计的有效活菌数目即为芽孢数。
3.芽孢率测定方法
芽孢率＝芽孢数/活菌数×100％
4.测定结果如表13
表13.测定结果
5.绝对货架期
实验方法：样品置于25℃下储存，定期进行稀释涂板，检测绝对货架期。存活率＝储存后芽孢数/初始(0M)芽孢数×100％。实验结果如表14所示。
表14.不同稀释倍数下的测定结果

实验结果表明，M173为产芽孢细菌，在菌体内形成圆形或椭圆形的芽孢休眠体。芽孢含水量极低，抗逆性强，能经受高温、紫外线，电离辐射以及多种化学物质灭杀等，芽孢率指标与活菌产品货架期呈正相关；测试结果显示本发明的M173存活率能稳定地达到90％以上，芽孢数也达到7.5×109cfu/mL；以上综合表明，M173具有芽孢数稳定、保持活菌数维持高的特点、以及货架期长的特性，表明M173具备上市成为产品的品质特性。
(6)M173的代谢产物测定
1、激素测定
M173激素测定方法：1)制作样品：将M173样品加入5ml 50％甲醇/水溶解，混合均匀后，吸取100μl样品溶液进行过0.22μm滤膜，最后在液相小瓶中定容至0.5ml。2)对照：IAA、IBA、茉莉酸甲酯、赤霉素、水杨酸甲酯、异戊烯基腺嘌呤核苷生长激素混合标准品。
测试仪器：使用LC-QQQ(三重串联四极杆液质联用仪)检验样品中所含生长激素的含量。
2、氨基酸测定
M173氨基酸含量测定方法
1)取1mL待水解样品加入600μL 6M的盐酸，110℃水解16h；
2)水解完成后，进行旋蒸浓缩；
3)用1mL甲醇复溶，过0.22微孔滤膜处理；
4)将过滤后的样品进行LC-QQQ定量分析，根据氨基酸标准曲线，计算样品中氨基酸含量。
激素和氨基酸测定的结果如表15和16所示。其中，表15表明M173菌株能够分泌激素IP、GA3、IPA、IAA、Me-SA、SA、IBA。表明M173可以促进植物的生长，促进植物生根，以及是的一部分植物受到病原体感染时，通过本发明的菌株M173可以促进植物对病原体产生抗性，进而提示其具有防病害的用途。表16表明M173产多种氨基酸，而氨基酸能提升作物品质、增强作物新陈代谢，促进作物生长。
表15.激素测定结果
表16.氨基酸测定结果(每毫升样品含量(mg/mL))
(7)M173的活性物质
首先，利用安捷伦LC-QTOF高分辨质谱对样品提取物进行二级质谱采集；其次，将原始数据文件进行格式转化；然后，上传至Massive质谱数据存储平台；最后，利用相应的GNPS(全球天然产物社会数据库)算法建立可视化的分子网络，识别已知多肽及其结构类似物。
实验结果证实，本发明的菌株可产生如下1.1至1.3所示的系列环肽类化合物，帮助作物抵抗真、细菌病害侵染。其中，环二、环三肽属于结构稳定且具有多种生物活性的化合物，芽孢杆菌霉素(bacillomycin)具有很强的广谱抑制病原真菌的效果和以及具抑制病原细菌的活性，表面活性素(surfactin)除具有抗病毒，还可抗细菌、抗真菌，抗菌谱较广，同时具有不易产生耐药性、可被动物消化酶降解、无残留等优点。
1.1环二、环三肽
1.2芽孢杆菌霉素
1.3表面活性素
(8)M173耐酸碱，耐高盐实验
1.1实验方法：
耐酸碱测试：配制LB液体培养基，分别调节培养基的pH值至3/5/7/9/11(用1mol/L的HCl和1mol/L的NaOH进行调节)，121℃20min高温高压灭菌。
将M173从甘油管活化至LB培养基固体平板，30℃培养箱下培养1-2d，将M173接种至LB液体培养基，摇床培养24h作为种子液，按照每瓶1mL的接种量将M173接种至不同pH值的LB培养基，3次重复，摇床恒温30℃下培养，培养至24h和48h时，取样测量OD600值并稀释涂板统计活菌数。
耐盐性测试：分别配制含NaCl 0.05mol/L、0.1mol/L、0.15mol/L、0.2mol/L、0.25mol/L的LB培养基，121℃20min高温高压灭菌。
将M173的种子液按照每瓶1mL的接种量接种至不同含盐量的LB培养基，以不加NaCl的培养基为对照，3次重复，摇床恒温30℃下培养，培养至24h和48h时，取样测量OD600值并稀释涂板统计活菌数。
1.2结果：
不同pH值环境对贝莱斯芽孢杆菌M173生长的影响见图8和表17，实验结果表明：M173在不同pH环境下均能生长繁殖，24h时pH3菌数较少，pH9的菌数显著大于pH7，但48h后除pH3外各处理菌数和pH7无显著差异，表明M173耐受pH范围较大。
综上所述，M173可以在PH3～PH11的环境下生长，在极度酸性和碱性环境下都能生长，且维持较高的活菌数量。
表17：不同pH对贝莱斯芽孢杆菌M173生长的影响

注：字母不同代表差异性显著，P<0.05
不同盐份浓度环境对贝莱斯芽孢杆菌M173生长的影响见下图9和表18，实验结果表明：M173的生长繁殖不受盐份浓度的影响，其中24h时0.3％的含盐量对M173的生长繁殖具有促进作用。高盐浓度1.5％环境对M173的生长仍无显著抑制，说明M173具有很好的耐盐性。
参照表19，M173可以在各种土壤环境中生长繁殖，尤其能够在强酸性土壤(pH＜5.0)和重盐碱地(pH＞9.5，含盐量＞0.6％)上生长繁殖。
表18：不同NaCl含量对贝莱斯芽孢杆菌M173生长的影响

注：字母不同代表差异性显著，P<0.05
表19：不同酸碱土壤的含盐量和pH范围划分


注：字母不同代表差异性显著，P<0.05
实施例12.M173安全性实验
1.实验方法：
番茄栽培与处理
育苗：将番茄每盆种植2棵，每盘6盆为1个处理，共12株苗，每处理3重复，共36株苗。水肥管理：除按时浇灌菌剂外，中间出现干旱情况浇0.01％NPK水溶肥。菌剂处理：对12天苗进行第1次浇灌，间隔7天进行第2次浇灌，共浇灌2次。
菌剂制备
菌剂稀释：用自来水将所需菌剂稀释至所需的倍数，如1L/亩的处理，按照亩2000株计算，即单株0.5mL。每盆2株苗共需菌剂1mL，浇灌50mL溶液，则该处理的稀释倍数为50。
2.数据收集
第2次浇灌后继续培养1周，收苗。选择记录番茄苗的株高、茎粗和干重等生理数据，进行处理分析。以生物量增加10％为促生标准。
株高：植株的高度(从根颈部到生长点的距离)。茎粗：以第一片真叶以下为基准，测定植株茎直径。
结果如表20所示。实验结果显示，M173的1L-4L/亩用量处理对番茄皆有促生效果，干重增长率在14.54％-61.49％之间，用量越多促生效果越好。因此，本发明的M173对番茄安全，且安全系数较高。
表20：各处理对番茄生长指标影响


注：字母不同代表差异性显著，P<0.05
实施例13.M173以及M173与腐殖酸复配的促生能力
1、试验材料与处理：
黄瓜品种：粤秀三号，广东科农生物科技
供试产品：M173菌株；根罗(FMC腐殖酸产品)；卓润(竞品)。
表21：各处理组内容
2、试验方法：
2.1黄瓜栽培与处理
育苗：将黄瓜种子每盆播种3颗，待长至12d间苗至每盆2株，每盘6盆为1个处理，每处理3重复，共36株苗。
水肥管理：适量浇灌0.01％NPK水溶肥，以保证黄瓜苗正常生长。
2.2菌剂处理
按照表21用自来水将各产品进行不同倍数的稀释，每盆浇灌50mL。
2.3数据收集
继续培养11d，收苗。选择记录黄瓜苗的展开度、茎粗和干重等生理数据，进行处理分析。
展开度：黄瓜2片真叶叶尖的距离。
茎粗：以第一片真叶以下为基准，测定植株茎直径。
实验结果(表22和图10)：单独施用的情况下，M173对黄瓜的促生效果优于根罗和卓润；在此基础上，M173和根罗复配促生效果优于单独使用，M173亩用量1L对黄瓜促生效果较好，干重增长率121.93％。根罗和卓润也有较好的促生效果，干重增长率在80％以上。M173和根罗以亩用量各1L复配的促生效 果最好，干重增长率160.03％。该处理的黄瓜展开度和茎粗也表现最好。
表22：各处理对黄瓜生长指标的影响

注：字母不同代表差异性显著，P<0.05
实施例14.M173与化药兼容实验
购买市售成品化药，将这些化药按照说明书稀释成相应浓度，M173菌液按200倍稀释加入至稀释好的化药溶液中，将化药和菌剂混合均匀后静置，分别在0、2、6h后取样测试菌数，计算存活率(注：存活率是由菌液稀释涂板的方法统计的，而稀释涂板可能存在一定的系统误差，因此个别存活率会大于100％；若存活率大于100％可以理解为M173与化药的兼容性非常好)。
存活率＝混合后Nh菌数/初始(0h)芽孢数×100％；其中，Nh代表第几小时，N取数值2或6；
实验结果如表23和24所示，结果显示，本发明的菌株M173与市面上常见的杀菌剂、杀虫剂和化肥均兼容。
表23：M173与化药兼容实验

表24：与化药兼容实验总结
实施例15.M173提高作物抗逆性实验报告
1.耐盐(NaCl)盆栽实验
1)耐盐测试实验方法：
原理：植物由细胞构成，细胞的稳定主要通过细胞内外离子浓度差维持，当植物叶片中的Na+浓度较高时，植物叶尖会出现卷曲或者叶边缘出现焦枯的现象。Na+浓度较高还会使植物对Ca2+的吸收受阻，造成植物的缺钙现象，出现衰苗、不结果的现象受阻的钙离子积累连带导致系列作物元素吸收受阻。
将育好的黄瓜苗间苗至每盆2株苗，每个处理6盆，3次重复。试验组设置清水空白对照(UTC)和浇灌复合菌剂2种处理，对12d的黄瓜苗进行浇灌(每株1mL，稀释50倍后浇灌)，UTC浇灌等量清水。3d后进行盐胁迫，采用向土壤基质中浇灌相应浓度盐溶液的方法。设置3个氯化钠溶液梯度(0、100、200mM)，每盆浇灌50mL。以后每隔3d浇灌一次盐溶液，14d后测定各指标。以干重等生长数据为主。
2)耐盐测试结果
浇灌了M173的黄瓜苗整体长势较未处理组(UTC)(表25和图11)，生物量均较UTC组高，随着氯化钠浓度的升高，黄瓜干重呈下降趋势。M173处理 的黄瓜生物量在不同盐浓度处理下均较UTC组高，干重增长率在10％以上，且随着盐浓度的升高，浇灌了M173的黄瓜生物量增长率越高，200mM NaCl处理的条件下，M173处理的黄瓜生物量较UTC增加了19.19％。
表25：耐盐试验各处理的干重及增长率

注：字母不同代表差异性显著，P<0.05
浇灌100mM氯化钠处理对黄瓜的盐害较轻，UTC和M173的2种处理的黄瓜均没有明显的盐害反应。浇灌200mM氯化钠处理的UTC组黄瓜出现了叶片干枯、植株死亡的现象，浇灌了M173的黄瓜植株仍然健康生长，没有盐害现象(图12和图13)。
3)实验结论：M173在正常条件和盐胁迫下均可以促进黄瓜生长，M173可以降低高盐环境对黄瓜造成的伤害。M173处理的黄瓜在盐胁迫下生长较好，有减轻作物盐害的效果，为其在含盐量较高的不良土壤环境下的推广提供了理论依据。
实施例16.解药害测试盆栽实验
1)实验方法：精甲霜·锰锌是由精甲霜灵与代森锰锌按科学比例混配的一种低毒复合杀菌剂，专用于防治低等真菌性病害，具有保护和治疗双重作用，两种杀菌机制，优势互补，可延缓病菌产生抗药性，使用安全。
生产中主要用于防治:西红柿晚疫病，黄瓜、甜瓜等瓜类霜霉病，辣椒的疫病、霜霉病，花椰菜霜霉病，西瓜及甜瓜的疫病，马铃薯晩疫病，烟草黑胫病，葡萄霜霉病，荔枝霜疫霉病等。
将育好的黄瓜苗间苗至每盆2株苗，每个处理6盆，3次重复。试验组设置清水空白对照(CK)和浇灌菌剂2种处理，对长出真叶的黄瓜苗进行喷施化药精甲霜·锰锌，设置500倍(推荐稀释倍数)、250倍(2×)、125倍(4×)的梯度，均匀喷施在黄瓜表面，4-7d后(视药害情形)浇灌菌剂(每株1mL，稀释50倍后浇灌)，CK浇灌等量清水。7d后测定各指标。以株高、茎粗、干重等生长数据为主，同时记录药害等级。
药害分级标准：
0级：黄瓜苗叶片正常
1级：黄瓜苗叶片黄化和斑点面积小于1/4
2级：黄瓜苗叶片黄化和斑点面积在1/4～1/2之间
3级：黄瓜苗叶片黄化和斑点面积大于1/2，但未占满整个叶片
4级：黄瓜苗叶片完全黄化干枯
药害指数＝(∑(各级叶数×各级代表数值)/(调查总叶片数×发病最高级数值))×100
相对防效(％)＝(对照组药害指数-处理组药害指数)/对照组药害指数×100％
表26：药害测试各处理内容
2)实验结果(图14)：喷施了精甲霜·锰锌的黄瓜出现了不同程度的药害现象，主要表现在叶片黄化、有干枯的斑点，严重的表现为叶片干枯卷曲。整体呈现浓度越高药害现象越严重的趋势。推荐稀释倍数(500倍)处理的黄瓜有少量叶片黄化，可能与黄瓜苗较小有关。
浇灌了M173的黄瓜苗药害情况均较UTC组有所减轻。高浓度(125倍稀释)化学农药处理下，M173处理组和UTC组药害指数具有显著差异，M173处理的药害防效达47.18％(表27)。250倍稀释化药和500倍稀释化药喷施的M173也有一定的防效，分别为21.32％和15.01％(表27)。
UTC组和M173处理组的展开度和茎粗之间没有显著差异，但数值上M173处理有一定优势。中浓度(250倍稀释)精甲霜·锰锌喷施的黄瓜，M173处理的生物量较UTC组提高了19.51％，且差异显著。
表27：解药害试验的生理指标与药害指数


注：字母不同代表差异性显著，P<0.05
3)实验结论：M173可以缓解精甲霜·锰锌的药害。在高浓度(125倍稀释)化药喷施条件下，M173处理的黄瓜的药害防效达47.18％。
实施例17.抗寒测试盆栽实验
1)实验方法：将育好的黄瓜苗间苗至每盆2株苗，每个处理6盆，3次重复。试验组设置如表28共6个处理，其中，竞品为微生物菌剂岱波路。测试条件为常温25℃/20℃(day/night)，低温15℃/10℃(day/night)，根系湿度皆保持70～90％；光照强度相等，在700～1200μmol/s。对12d的黄瓜苗进行菌剂浇灌，对照浇灌等量清水，7d后转移至低温培养箱培养14d后测定各指标。以株高、茎粗、叶绿素含量、干重等生长数据为主，若差异明显视情况测定丙二醛等膜脂过氧化指标(马俊，2016)。
表28：抗寒测试各处理组内容
2)实验分析及实验结果：
实验结果如图15和表29所示。低温胁迫在植物生长发育的各阶段都会产生不良影响，在本试验中，低温处理的黄瓜苗长势较正常温度条件(UTC)下的差，叶片发黄，叶绿素含量下降。M173处理的黄瓜在低温和正常条件下展开度和叶绿素含量均高于CK和竞品(岱波路)。M173处理的单株干重在正常温度下较CK增加了16.22％，在低温条件下较CK增加了10.96％，均具有较好的促生效果。竞品(岱波路)在低温和正常条件下均没有促生效果。
低温逆境条件下，M173可以通过促进作物生长、提高叶绿素含量等方式来降低 逆境的不良影响。
表29：抗寒试验的生理指标

注：字母不同代表差异性显著，P<0.05
3)实验结论：M173可以促进黄瓜在低温环境下的生长，降低低温逆境对黄瓜造成的伤害。
实施例18.抗旱测试盆栽实验
1)实验方法：
原理：PEG-6000(聚乙二醇)是一种理想的高分子渗透剂，渗透压稳定，限制植物水分吸收速率，可以用于干旱缺水环境的模拟。
选取饱满、大小一致的黄瓜种子播种于育苗盘，定期定量浇灌。将12d左右的黄瓜苗移栽至无孔种植盆，每盆1株苗，每个处理6盆苗，3次重复。试验组设置清水空白对照(CK-UTC)、20％PEG-6000处理(CK-PEG)以及在PEG处理的基础上浇灌复合菌剂。移栽后先浇灌复合菌剂(每株1mL，稀释50倍后浇灌，以浇透为宜)，CK-UTC和CK-PEG处理浇灌等量清水。缓苗后向除CK-UTC外的幼苗浇灌20％PEG-6000一次，保证土壤浸透溶液。将处理好的盆栽置于温室培养，定期观察。12d后统计幼苗存活率、视情况记录茎粗、干重及展开度。
表30：抗旱测试各处理内容
2)实验分析及实验结果
实验结果如图16和表31所示。干旱胁迫下植物根系吸水受阻，影响植物的正常 生长发育。在本试验中，CK-PEG处理的黄瓜苗长势较正常温度条件(CK-UTC)下的差，展开度、茎粗和单株干重均较CK-UTC下降，且2个处理的展开度和茎粗间差异显著。
PEG模拟的干旱条件下，浇灌了M173的黄瓜苗长势最好，展开度较CK处理显著提高，茎粗和单株干重也表现最好，甚至已经接近未进行干旱处理的黄瓜长势。
表31：抗旱试验的生理指标

注：字母不同代表差异性显著，P<0.05
3)实验结果：M173可以提高干旱环境下黄瓜的长势，减轻干旱胁迫对作物生长的抑制，表明干旱逆境条件下，M173可以通过促进作物生长来降低逆境的不良影响。
实施例19.平板抑制病原菌实验
1.1供试病原菌：灰霉菌(Botrytis cinerea)、炭疽菌(Colletotrichum capsici)、尖孢镰刀菌(Fusarium oxysporum)、立枯丝核菌(Rhizoctonia solani)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、青枯菌(Ralstonia solanacearum)。
1.2分组：分为空白对照组，对照贝莱斯芽孢杆菌组(这些菌株是参照实施例1记载的筛选方法和鉴定方法获得的)和贝莱斯芽孢杆菌M173组，每组5个重复。
1.3试验方法：
空白对照组：(真菌的处理)从活化3天的真菌病原菌菌落边缘打取菌饼(直径5mm)，将其置于离PDA平板中心2.5cm处，28℃恒温培养。(细菌的处理)吸取疮痂链霉菌、青枯菌病原菌菌液100μL至LA平板上，均匀涂布并晾干。
对照贝莱斯芽孢杆菌组(贝莱斯芽孢杆菌1、贝莱斯芽孢杆菌2、贝莱斯芽孢杆菌3、贝莱斯芽孢杆菌4、贝莱斯芽孢杆菌5、贝莱斯芽孢杆菌6，这些菌株均是按照实施例1记载的筛选方法和鉴定方法获得的)；贝莱斯芽孢杆菌M173组：(抑真菌测试)从活化3天的真菌病原菌菌落边缘打取菌饼(直径5mm)，将其置于PDA 平板中央，刮取活化1天的贝莱斯芽孢杆菌M173菌苔，在病原菌菌饼两侧2cm处平行划直线接种，28℃恒温培养。(抑细菌测试)吸取疮痂链霉菌、青枯菌病原菌菌液100μL至LA平板上，均匀涂布并晾干，用打孔机将滤纸打出直径为0.6cm的纸片，灭菌备用；将无菌的滤纸片用镊子置于LA平板上，每板3片，呈正三角分布，吸取5μl M173的发酵液滴于上述平板的滤纸片上。
1.4抑制率计算：
接种后，平板倒置培养，1-7d后测量病原菌直径/抑菌圈宽度，并根据以下公式计算抑菌率。抑菌率计算公式如下：
真菌抑菌率＝(空白对照病原菌菌落直径-贝莱斯芽孢杆菌M173处理的病原菌菌落直径)/空白对照病原菌菌落直径×100％；
细菌抑菌圈宽度＝(抑菌圈直径-贝莱斯芽孢杆菌菌体直径)/2；
细菌拮抗指数＝抑菌圈宽度/贝莱斯芽孢杆菌菌体半径。
1.5结果：
表32：贝莱斯芽孢杆菌M173抑菌实验结果
由表32和图17可得，贝莱斯芽孢杆菌M173对真菌性病原菌和细菌性病原菌有较好的抑制作用。
对于真菌性病害，本发明的菌株贝莱斯芽孢杆菌M173菌在农作物抗菌，尤其是抗灰霉病、枯萎病、立枯病、白绢病等方面有较好的应用前景。
对于细菌性病害，平板对峙实验可以看出本发明的菌株M173对于细菌性病害(马铃薯疮痂病、青枯病)有较好的抑制效果。
表33：贝莱斯芽孢杆菌的筛选：

表33的实验结果显示，即便同样属于贝莱斯芽孢杆菌(Bacillus velezensis)，不同菌株的特性以及生防效果有着非常大的区别。在这其中，本发明的M173菌株具有突出的优势。
实施例20.贝莱斯芽孢杆菌花生种衣剂
1.1花生包衣
常规种衣剂制备：常规种衣剂配方：蔗糖5％，PVPK30 10％，PEG 3％，分散剂MF 2％，聚乙二醇5％。各组分按照质量分数进行配比。配制10亿CFU/mL的种衣剂，将大小、质地相似的花生种子以1：100(微生物种衣剂与种子重量比)的量加入到微生物种衣剂中，震荡使菌株均匀吸附于种子上，通过M173包衣花生对其萌发以及苗期生长的影响测试。
1.2种子包衣处理后表面菌数
表34：各处理包衣种子表面菌数
1.3包衣后种子发芽率
表35：各处理对花生发芽率的影响
表35的结果证实，M173包衣对花生的发芽有一定促进作用，发芽率为76.67％， 显著高于空白种衣剂组51.67％，高于CK组63.33％。
1.4包衣对花生促生指标的影响
如图18，图19和表36所示，M173包衣后的花生长势最好，株高、地上部干重、地下部干重均表现最好，但与CK无显著差异。
表36：各处理对花生生长指标的影响

注：字母不同代表差异性显著，P<0.05
实施例21.M173种子包衣防治玉米茎腐病实验
1.1实验材料与实验组处理：
玉米品种：金玉509
菌剂样品：M173。
病原菌的制备：禾谷镰刀菌(P067)，CMC培养基培养至10^6cfu/ml(摇瓶培养7天)。
化药对照：咯菌·精甲霜悬浮种衣剂。
表37：药剂用量
1.2实验方法
常规种衣剂制备：常规种衣剂配方：蔗糖5％，PVPK30 10％，PEG 3％，分散剂MF 2％，聚乙二醇5％。各组分按照质量分数进行配比。配制10亿CFU/mL的种衣剂，将大小、质地相似的玉米种子以1：100(微生物种衣剂与种子重量比)的量加入到微生物种衣剂中，震荡使菌株均匀吸附于种子上，通过M173包衣玉米对其萌发以及苗期生长的影响测试。
助剂对照为种衣剂中的微生物部分填充为麦芽糊精的空白种衣剂；化药为咯菌·精甲霜，稀释10倍，1:80包衣。
将拌种后的种子，晾干后种植于灭菌土中，3棵/盆，6盆/重复，3重复/处理。将种植后的玉米浇灌病原菌10^6cfu/ml，50ml/盆。每天加水保湿，使土壤湿度一直维持在90％以上。
水肥管理：第七天浇1‰NPK水溶肥。
1.3实验数据
种植2周后收苗。记录各处理玉米的病情指数。
玉米苗期茎基腐病病情指数分级：
0级：整株生长正常，无病。
1级：地上地下部分基本正常，根部可见少量病斑，病斑面积占根表面积1/4以下，根群颜色白中有褐。
2级：地上地下生长明显受阻，叶色变黄，株高仅及对照的3/4，侧根少而短，无须根，病斑连片，病斑面积占根表总面积的1/4-1/2，根群颜色白、褐相当。
3级：地上地下部分生长极不正常，地上部可见黄枯状，株高仅为对照的1/2，侧根极小，病斑面积占根总面积的1/2-3/4，根群颜色褐中带白。
4级：发芽，但不出苗，几乎窒息而死，病斑面积占根表总面积的3/4以上，根为褐色。
病情指数(％)＝(∑(每个病级的植株数×级别数)/(总植株数×最高级别数))×100％。
相对防效(％)＝((阴性对照组病情指数-实验组病情指数)/阴性对照组病情指数)×100％。
表38：各处理的玉米发芽率、病情指数和相对防效

注：字母不同代表差异性显著，P<0.05
实施例22.M173番茄青枯灌根实验
1.1实验材料
番茄品种：新星101，广东科农生物科技；
慕恩产品：M173菌剂(菌数为100亿cfu/mL)；竞品：卓润。
青枯劳尔氏菌R.s(Ralstonia solanacearum)，该病原菌菌株是从华农试验田中发病番茄植株上分离出的具有高致病性的菌株，其编号为MN R.S2。
1.2 R.s菌悬液制备
将青枯劳尔氏菌R.s(Ralstonia solanacearum)从甘油管中活化到TTC平板(配方为：葡萄糖5g，蛋白胨10g，水解络蛋白1g，琼脂15g，加水至1L，121℃灭菌20min，冷却至60℃后加TTC，使终浓度为0.005％(W/V))上，培养2d后选取活性高的青枯菌落转接至SPA液体培养基(配方为：蔗糖20g，细菌学蛋白胨5g，K2HPO4 0.5g，MgSO4 0.25g，加水至1L，调至pH为7.0～7.2，121℃灭菌20min)中，摇床上培养8-16h后再根据OD600的吸光度值将R.s稀释至3*106CFU/mL，制得R.s菌悬液备用。
1.3 M173的发酵和活化
对M173菌株的发酵过程进行优化，选择最优配比的碳源和氮源并控制PH环境，以使得M173能够发酵到菌数为100亿cfu/mL。
具体来说，优化的培养基为：碳源30～50g/L(淀粉水解液、玉米粉、葡萄糖或蔗糖选其一)、氮源40～60g/L(豆粕或蛋白胨选其一)、无机盐(包括磷酸氢二钠2～5g/L和硫酸镁0.5～2g/L和碳酸钠1～2g/L)、微量元素0.01-0.02g/L(ZnSO4和/或MnSO4)；
发酵工艺为：以50L发酵罐为例，发酵开始时pH用2M稀硫酸调至7±0.2，并在发酵过程中控制pH在6-8之间；发酵前期提高溶氧，风量0.5-1.2vvm，转速500-600rpm；发酵后期控制溶氧，风量0.2-0.5vvm，转速200-300rpm。
1.4产品稀释
将处理液按照表39用清水稀释。
表39：试验处理表

1.5番茄苗处理
将16d番茄苗，轻轻抖去根表面泥土，随即种入未灭菌土中，每盆1株，10盆为1个处理，3重复。根据处理液不同设置阴性对照组、阳性对照组和菌剂处理组。阴性对照组的处理液为清水，阳性对照组的处理液为可杀得1000倍稀释液，菌剂处理组的处理液为4.2制备的生防菌剂。每盆浇灌50mL相对应的处理液，将盆栽置于25-28℃环境中缓苗7d。7d后将各个处理组的每株苗灌入50mL4.2制备的R.s菌悬液(阳性对照组将菌悬液中加适量可杀得颗粒剂，使浇灌的终浓度为1000倍)，将番茄苗移至32℃温室发病棚中(不密闭)促使发病。
1.6观察记录：
在出现症状的第一个迹象(番茄苗出现青枯症状)开始(一般为灌根后第3-4d开始发病)，每1天或2天观测记录一次番茄苗状态(根据分级标准记录发病情况)，至第7d-13d(视实际发病情况确定)，记录各处理每个病级的植株数，根据以下公式
计算发病率和病情指数。
植株病情等级分为以下5级：
0级：无症状
1级：一片叶萎蔫
2级：2-3片叶萎蔫
3级：整株枯萎
4级：植株死亡
病情指数(％)＝(∑(每个病级的植株数×级别数)/(总植株数×最高级别数))×100％
防治效果(％)＝((阴性对照组病情指数-实验组病情指数)/阴性对照组病情指数)×100％
1.7实验结果
表40：各不同处理下番茄的发病情况


注：字母不同代表差异性显著，P<0.05。
本批次试验CK的发病率和病情指数均在90％以上，发病严重，有利于评价生防菌的效果。
M173对番茄青枯病的防治效果随用量增加而提高，1L/亩对番茄青枯病的防效为53.64％，2L/亩防效77.27％。M173的促生效果较好，2L/亩的干重增长率达57.72％。竞品卓润的防效较为一般，为12.73％，化药可杀得的防效为41.72％。
实施例23.贝莱斯芽孢杆菌M173田间试验
本实施例测试了M173防死棵的效果，具体方法如表41所示。
表41：试验设置
1.M173防治苦瓜死棵促生长
本实施例提供了贝莱斯芽孢杆菌M173用于苦瓜试验田的案例。
试验田地址:广东省南沙区六顷东。
试验田基本情况:试验地选在大片苦瓜靠近地头区域，往年种植番茄出现较多死棵，试验地选择3垄，1垄设置1个重复，1个重复内土肥条件、光照、灌溉、地势一致。
M173用于试验田的方法如下:
设置M173的梯度处理，单株0.5mL(400倍)和1mL(200倍)，以竞品卓润 (推荐用量单株0.5mL)为对照，化药处理选用3％甲霜·噁霉灵，单株用量0.33mL(600倍)，以未处理作为空白对照。采用随机区组设计，每个小区面积14平方米，3重复。生育期进行3次施药，间隔10-14d，采用稀释后灌根的处理方式，单株浇灌水量200mL。
在第一次药后14天调查株高(图20)，M173、卓润、甲霜·噁霉灵的株高较CK均有增长，以M173 200倍、卓润400倍效果优于其他处理，甲霜·噁霉灵促生长效果低于M173的400倍。
在第三次药后调查植株长势(图21)，长势对比：M173 200倍>甲霜·噁霉灵>M173400倍>卓润400倍>CK，从挂果数上来看：M173 200倍>M173 400倍>卓润400倍＝甲霜·噁霉灵>CK
综上：M173在苦瓜上具有明显的促生长效果，200倍的促生效果优于400倍。同等剂量下，M173在苦瓜上的促生长效果优于卓润，尤其是在挂果数量上明显优于卓润。
M173低剂量在苦瓜上的促生效果与甲霜·噁霉灵相当，但是M173在苦瓜挂果数量上明显多于甲霜·噁霉灵。
2.M173防治罗汉果死棵
罗汉果在缓苗期非常容易出现死棵问题，本实施例提供了贝莱斯芽孢杆菌M173用于罗汉果试验田的案例。
试验田地址:广东省清远市望埠镇。
试验田基本情况:试验地罗汉果种植面积达200亩。移栽前，前茬种植冬瓜，罗汉果在移栽后的缓苗期非常容易出现死棵问题，如何让罗汉果度过缓苗期是农户痛点。
M173用于试验田的方法如下:
设置M173的梯度处理，单株1mL(200倍)和2mL(100倍)，以竞品卓润(单株1mL)为对照，化药处理选用3％甲霜·噁霉灵，单株用量1mL，以未处理作为空白对照。采用随机区组设计，每个小区面积60平方米，3重复。生育期进行3次施药，间隔14-20d，采用稀释后灌根的处理方式，单株浇灌水量200mL。
统计每个小区从移栽开始正常生长的植株的数量、每个小区移栽后出现死棵，通过补苗生长起来的植株的数量。
正常植株占比：正常植株数/总植株数*100
成活率：总成活株数/总植株数*100
本次试验中，从移栽后第一次施药开始到最后一次调查结束，农户进行了大量补苗，从移栽后60天调查的正常植株占比、成活率可以反映出试验药剂在提高罗汉果移栽成活率、减少死棵方面的效果。
结果如图22所示，与CK对比：M173-1毫升/株能够增加罗汉果23％的正常植株占比，增加41％的成活率；M173-2毫升/株能够增加罗汉果20％的正常植株占比，增加39％的成活率；卓润1毫升/株能够增加罗汉果20％的正常植株占比，增加36％的成活率；3％甲霜·噁霉灵1毫升/株能够增加罗汉果14％的正常植株占比，增加33％的成活率；
效果对比：M173-1毫升/株(200倍)>M173-2毫升/株(100倍)>卓润1毫升/株>3％甲霜·噁霉灵1毫升/株。
实施例24.青枯病田间实验(辣椒青枯&番茄青枯)
1.1 M173防治辣椒青枯病
本实施例提供了贝莱斯芽孢杆菌M173用于辣椒试验田的案例。
试验田地址:广东省广州市增城试验田。
试验田基本情况:前茬作物为茄子，露天种植，2000株每亩。
M173用于试验田的方法如下:
设置M173的梯度处理，单株0.5mL(400倍)和1mL(200倍)，以竞品卓润(单株0.5mL)为对照，化药处理选用3％甲霜·噁霉灵，单株用量0.33mL(600倍)，以未处理作为空白对照。采用随机区组设计，每个小区面积14平方米，3重复。生育期进行4次施药，间隔7d，采用稀释后灌根的处理方式，单株浇灌水量200mL。
株高调查：随机选择10株调查株高；坐果数调查：数小区内总挂果数。每个小区有40株辣椒。
防效：(CK发病率-处理组发病率)/CK发病率×100％。
株高增长率分别为：173-2升/亩17.5％，173-1升/亩7.4％；卓润11.4％
173-2升/亩坐果数增加15.2％，173-1升/亩坐果数增加20％，卓润坐果数增加21.5％(173-2升/亩等同于单株0.5mL，173-1升/亩等同于1mL/亩，下同)。
实验结果如表42所示，173-1mL/株防效达53％，显著优于-0.5mL/株37％的防效，优于卓润的防治效果。
表42：M173防治辣椒青枯病
1.2 M173防治番茄青枯病
本实施例提供了贝莱斯芽孢杆菌M173用于番茄青枯试验田的案例。
试验田地址:广东省广州市南沙区横沥镇。
表43：
表44：

M173用于试验田的方法如下:
设置M173的梯度处理，单株0.5mL(400倍)和1mL(200倍)，以竞品阵地(单株1mL)为对照，化药处理选用20％噻菌铜和20％噻唑锌，单株用量分别为0.4mL(500倍)和0.5mL，以未处理作为空白对照。采用随机区组设计。生育期进行3次施药，间隔7d，采用稀释后灌根的处理方式，单株浇灌水量200mL。
防效＝(CK发病率-处理发病率)/CK发病率×100％
试验地1：
实验结果如图23所示，M173-2升/亩的防效显著高于1升/亩的防效。在CK发病率39％的情况下，M173-2升/亩对番茄青枯病的防效超过90％，M173-2升/亩的防效高于阵地2升/亩、20％噻菌铜0.8升/亩。M173-2升/亩的防高最高。
试验地2：
实验结果如图24所示，试验地发病较轻，只有3个小区发病，分别是CK、阵地2升/亩、20％噻唑锌1升/亩。
M173两个处理、20％噻菌铜的两个重复均未发病。
试验地3：
实验结果如图25所示，M173-2升/亩的防效显著高于1升/亩的防效。M173-2升/亩对番茄青枯病的防效高达70％以上。M173-1升/亩的防效高于阵地2升/亩、20％噻唑锌1升/亩，低于20％噻菌铜0.8升/亩。M173-2升/亩的防效最高最稳定。
实施例25.M173防治兰花茎腐病
本实施例提供了贝莱斯芽孢杆菌M173用于兰花的案例。
试验田地址：肇庆市四会市石狗镇；兰花品种：墨兰；试验田基本情况:试验地墨兰茎腐病病原菌为尖孢镰刀菌，苗盘携带病原菌，开展本试验之前已经发病，在本试验开展之前把每个小区的发病株挑出来并补充未发病株，每个小区有42盆墨兰，均为未发病状态。
M173用于试验田的方法如下：
设置M173的梯度处理，单株0.5mL(400倍稀释)和1mL(200倍稀释)，以竞品卓润(推荐用量单株0.5mL)为对照，化药处理选用3％甲霜·噁霉灵，单株用量0.33mL(600倍稀释)，以未处理作为空白对照。采用随机区组设计，每个小区42盆兰花，3重复。生育期进行4次施药，每次施药间隔14天)，采用稀释后灌根的处理方式，单株浇灌水量200mL。在8月30日第一次调查茎腐病发病情况，9月9日进行第二次调查，计算各小区的发病率及相对防效。
计算公式：发病率＝发病株数/调查株数×100％；防效＝(空白发病率-处理发病率)/空白发病率×100％。
第一次发病调查可知(图26、图27)，M173 200倍稀释、400倍稀释对兰花茎腐病的防效基本相当，无剂量差异，防效达50％以上；M173对兰花茎腐病的防效与卓润(54％)相当，低于3％甲霜·噁霉灵的防效(65％)。
第二次发病调查可知(图28、图29)，M173 200倍稀释、400倍稀释对兰花茎腐病的防效基本相当，无剂量差异，防效达40％以上；M173对兰花茎腐病的防效低于卓润(52％)和3％甲霜·噁霉灵防效(56％)。图30，图31，图32和图33分别为各组处理的(M173(400倍稀释)，M173(200倍稀释)，卓润，3％甲霜·噁霉灵)兰花生长情况。
M173在第三次施药后的防效达50％以上，第四次施药后药效所有下降，达40％以上。
由此可见，本发明的M173贝莱斯芽孢杆菌对兰花茎腐病有较好的生物防治效果。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (18)

  1. 一种贝莱斯芽孢杆菌M173(Bacillus velezensis)或其变体或其后代，所述贝莱斯芽孢杆菌M173保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61434。
  2. 权利要求1所述的贝莱斯芽孢杆菌M173或其变体或其后代，其中，所述变体或后代与所述贝莱斯芽孢杆菌M173具有相似或相同的功能；
    优选地，所述贝莱斯芽孢杆菌M173的变体或后代与所述贝莱斯芽孢杆菌M173具有相似或相同的生理生化特性；
    优选地，所述贝莱斯芽孢杆菌M173具有选自下列的一项或多项功能：
    (1)能够提高植物苗期(例如，苗期移栽)存活率；
    (2)能够促进植物各个阶段(例如，幼苗期，缓苗期，生长期)的生长(例如，增加株高，茎粗)；
    (3)能够增加植物结果的产量；
    (4)能够促进植物器官生长(例如，根，茎，叶)；
    (5)能够提高植物对逆境环境的抗性；
    (6)能够提高植物对病原菌的抗性；
    (7)(1)至(6)。
  3. 一种组合物，其包含贝莱斯芽孢杆菌M173或其变体或其后代；
    优选地，所述组合物还包含一种或多种另外的生物防治剂，一种或多种化学药物，或其任意组合；
    优选地，所述组合物还包含农业上或园艺上可接受的稀释剂，填充剂、溶剂、自发性促进剂、载体、乳化剂、分散剂、防腐剂、防冻剂、增稠剂、佐剂，或其任意组合。
  4. 权利要求3所述的组合物，其中，所述另外的生物防治剂选自：细菌，真菌(例如，酵母)，病毒，昆虫，线虫，或其任何组合；
    优选地，所述细菌选自芽孢杆菌属，乳酸杆菌属，双歧杆菌属，丙酸杆菌属， 链球菌属，乳球菌属，片球菌属，肠球菌属，葡萄球菌属，或其任何组合；
    优选地，所述芽孢杆菌属的细菌选自：贝莱斯芽孢杆菌(Bacillus velezensis)，枯草芽孢杆菌(Bacillus subtilis)，短小芽孢杆菌(Bacillus pumilus)，凝结芽孢杆菌(Bacillus coagulans)，或其任何组合；
    优选地，所述另外的生物防治剂是保藏编号为GDMCC No.61962的贝短小芽孢杆菌(Bacillus pumilus)，所述短小芽孢杆菌保藏于广东省微生物菌种保藏中心；
    优选地，所述组合物中所述贝莱斯芽孢杆菌和所述短小芽孢杆菌的混合体积比例为1:5至5：1(例如，1：5；1：4；1：3；1：2；1：1；2：1；3：1；4：1；5：1；)；
    优选地，所述组合物中贝莱斯芽孢杆菌和短小芽孢杆菌的混合体积比例为2:1；
    优选地，所述酵母选自酿酒酵母(Saccharomyces cerevisiae)，布拉氏酵母(Saccharomyces boulardii)，马克斯克鲁维酵母(Kluyveromyces marxianus)，或其任何组合。
  5. 权利要求3或4所述的组合物，其中，所述化学药物选自：除草剂，杀虫剂，抗菌剂(例如，真菌，细菌)，抗病毒剂，植物生长调节剂，抗生素，肥料，或其任意组合；
    优选地，所述抗菌剂选自霜霉威盐酸盐、多菌灵、氟啶胺、百菌清、五氯硝基苯、福美双、三乙膦酸铝、精甲霜灵、精甲霜灵咯菌腈，或其任意组合；
    优选地，所述杀虫剂选自噻虫嗪、呋虫胺、精高效氯氟氰菊酯、丙溴辛硫磷，或其任意组合；
    优选地，所述肥料选自腐殖酸类肥料(例如，根罗)尿素、硫酸铵、欢之田、安糖可力、磷酸二氢钾，或其任意组合；
    优选地，所述组合物为固体形式，液体形式，粉末形式，或其任意组合。
  6. 一种生物培养物，其包含权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物；
    优选地，所述生物培养物还包含固体或液体培养基，或所述培养基的组分；
    优选地，所述生物培养物还包含贝莱斯芽孢杆菌M173的活细胞；优选地，所述活细胞以孢子形式存在；更优选地，所述孢子存在于贝莱斯芽孢杆菌M173的液体培养基中；
    优选地，所述生物培养物包含贝莱斯芽孢杆菌M173的细胞和培养物的上清液；
    优选地，所述培养基包含碳源(例如，淀粉水解液、玉米粉、葡萄糖或蔗糖)，氮源(例如，豆粕，蛋白胨)，无机盐(例如，磷酸氢二钠，硫酸镁，碳酸钠)，微量元素(例如，ZnSO4，MnSO4)，或其任意组合。
  7. 权利要求6所述的生物培养物，所述培养物还包含贝莱斯芽孢杆菌M173的衍生物或其子代培养物；
    优选地，所述衍生物或子代培养物选自代谢产物(例如，环二肽，环三肽，芽孢杆菌霉素，表面活性素)，激素(例如，IP，GA3，IPA，IAA，Me-SA，SA，IBA)，酶，细胞结构成分，或其任何组合；
    优选地，所述培养物还包含提供营养的成分；
    优选地，所述提供营养的成分选自蛋白质，碳水化合物，酶，维生素，矿物质，氨基酸，或其任何组合。
  8. 一种植物促生剂，其包含权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物；
    优选地，所述植物促生剂还包含一种或多种另外的生物防治剂，一种或多种肥料，或其任意组合；
    优选地，所述肥料选自腐殖酸类肥料(例如，根罗)或氨基酸水溶肥。
  9. 一种用于防治植物病原性细菌和/或促进植物生长的生物防治剂，其包含权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物。
  10. 一种用于植物的生长基质，其包含权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂，或权利要求9所述的生物防治剂；
    优选地，所述生长基质还包含沙子，土壤，惰性颗粒基质(例如，蛭石)； 或其任意组合。
  11. 一种农药组合物，其包含权利要求1所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂，或权利要求9所述的生物防治剂；
    优选地，所述农药组合物还包括一种或多种生物防治剂和/或一种或多种化学药物(例如，农药)。
  12. 经权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂或权利要求9所述的生物防治剂或权利要求11所述的农药组合物处理过的植物、植物组织或植物器官；
    优选地，所述处理包含浸根、叶喷、喷雾、堆肥、浸种、包衣、大田漫灌、滴灌植物或植物器官，涂抹植物或植物器官，滴加植物或植物器官；
    优选地，所述植物器官包含根、茎、叶、花、果实和种子；
    优选地，所述植物组织包含分生组织，保护组织，基本组织，和输导组织；
    优选地，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科；
    优选地，所述植物具有选自下列的一项或多项特征：
    (1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
    (2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
    (3)所述豆科植物选自大豆，花生，或其任意组合；
    (4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
    (5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
    (6)所述兰科植物选自兰花；
    优选地，所述处理包括灌根，浸种，种子包衣。
  13. 一种提高植物苗期(例如，缓苗期移栽)存活率的方法，所述方法包括：将 权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂或权利要求9所述的生物防治剂或权利要求11所述的农药组合物处理所述植物；
    优选地，所述处理选自灌根，浸根、叶喷、喷雾、堆肥、浸种、种子包衣、大田漫灌、滴灌；
    优选地，所述贝莱斯芽孢杆菌M173是通过选自下列的一项或多项特征来提高植物苗期存活率：
    (1)提高植物苗期移栽存活率；
    (2)促进植物生长(例如，增加株高，茎粗)；
    (3)增加植物结果的产量；
    (4)促进植物器官生长(例如，根，茎，叶)；
    (5)提高植物对逆境环境的抗性；
    (6)提高植物对病原菌的抗性；
    (7)(1)至(6)；
    优选地，所述逆境条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合；
    优选地，所述病原菌选自病原细菌，病原真菌或病毒；
    优选地，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌 (Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合；
    优选地，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)。
  14. 权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂或权利要求9所述的生物防治剂或权利要求11所述的农药组合物在预防和/或治疗由病原菌引起的植物病害中的用途，或者在预防和/或缓解由非病原菌条件引起的植物损伤或坏死中的用途；
    优选地，所述病原菌选自病原细菌，病原真菌或病毒；
    优选地，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌 (Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合；
    优选地，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)；
    优选地，所述非病原菌条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合；
    优选地，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科；
    优选地，所述植物具有选自下列的一项或多项特征：
    (1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
    (2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
    (3)所述豆科植物选自大豆，花生，或其任意组合；
    (4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
    (5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
    (6)所述兰科植物选自兰花。
  15. 权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂或权利要求9所述的生物防治剂或权利要求11所述的农药组合物在提高植物对病原菌或逆境条件的抗性的中的用途，或者在促进植物生长中的用途；
    优选地，所述病原菌选自病原细菌，病原真菌或病毒；
    优选地，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis  cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合；
    优选地，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)；
    优选地，所述逆境条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合；
    优选地，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科；
    优选地，所述植物具有选自下列的一项或多项特征：
    (1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
    (2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
    (3)所述豆科植物选自大豆，花生，或其任意组合；
    (4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果， 或其任意组合；
    (5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
    (6)所述兰科植物选自兰花。
  16. 一种预防和/或治疗由病原菌引起的植物病害，或者预防和/或缓解由非病原菌条件引起的植物损伤或坏死的方法，所述方法包括：将权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂，或权利要求9所述的生物防治剂，或权利要求11所述的农药组合物施用于植物、植物组织或植物器官；
    优选地，所述植物器官包含根、茎、叶、花、果实和种子；
    优选地，所述施用包含浸根、叶喷、喷雾、堆肥、浸种、包衣、大田漫灌、滴灌植物或植物器官，涂抹植物或植物器官，滴加植物或植物器官；
    优选地，所述植物组织包含分生组织，保护组织，基本组织，和输导组织；
    优选地，所述病原菌选自病原细菌，病原真菌或病毒；
    优选地，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌 (Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合；
    优选地，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)；
    优选地，所述非病原菌条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合；
    优选地，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科；
    优选地，所述植物具有选自下列的一项或多项特征：
    (1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
    (2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
    (3)所述豆科植物选自大豆，花生，或其任意组合；
    (4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
    (5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
    (6)所述兰科植物选自兰花。
  17. 一种提高植物对病原菌或逆境条件的抗性，或者促进植物生长的方法，所述方法包括：将权利要求1或2所述的贝莱斯芽孢杆菌M173或其变体或其后代，或者权利要求3-5任一项所述的组合物，或者权利要求6或7所述的生物培养物，或权利要求8所述的植物促生剂或权利要求9所述的生物防治剂或权利要求11所述的农药组合物施用于植物、植物组织或植物器官；
    优选地，所述植物器官包含根、茎、叶、花、果实和种子；
    优选地，所述施用包含浸根、叶喷、喷雾、堆肥、浸种、包衣、大田漫灌、滴灌植物或植物器官，涂抹植物或植物器官，滴加植物或植物器官；
    优选地，所述植物组织包含分生组织，保护组织，基本组织，和输导组织；
    优选地，所述施用包括：灌根，浸种，种子包衣；
    优选地，所述病原菌选自病原细菌，病原真菌或病毒；
    优选地，所述病原菌选自炭疽菌(Colletotrichum capsici)灰霉菌(Botrytis cinerea)，青枯劳尔氏菌(Ralstonia solanacearum)、立枯丝核菌(Rhizoctonia solani)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、罗耳阿太菌(Athelia rolfsii)、疮痂链霉菌(Streptomyces scabies)、花生白绢病菌(Sclerotium rolfsii)、油菜菌核病菌(Sclerotiniasclerotiorum)、黄瓜枯萎病菌(Fusariumoxysporum.sp.cucumebrium Owen)、小麦全蚀病菌(Gaeumannomycescritici)、小麦赤霉病菌(Fusariumgraminearum)、苹果树腐烂病菌(Valsamali)、苹果炭疽病菌(Glomerellacingulata)、水稻纹枯病菌(Rhizoctoniasolan)、稻瘟病菌(Pyriculariagrisea)、番茄早疫病菌(Alternariasolani)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)、猕猴桃溃疡病菌(Pseudomonas syringae)、水稻白叶枯病菌(Xanthomonascampestris)、白菜软腐病菌(Erwiniacarotorora)、核桃黑斑病菌(Xanthomonascampestris)和/或魔芋软腐病菌(Erwiniacarotovora)、草莓灰霉病菌(Botrytis cirerea)、马铃薯晚疫病菌(Phytophthorainfestans)、玉米大斑病菌(Exserohilumturcicum)、玉米小斑病菌(Bipolariamaydis)、西瓜枯萎病菌(Fusariumoxysporumf.sp.niveum)、茄子黄萎病菌(Verticilliumdahliae)、棉花枯萎病菌(Fusariumoxysporumf.sp.vasinfectum)、辣椒疫霉病菌(Phytophthoracapsici)、烟草疫霉病菌(Phytophthoranicotianae)，或其任意组合；
    优选地，所述病原菌引起的植物病害选自植物的青枯病(例如，茄科青枯病，例如，辣椒青枯病，番茄青枯病)，植物的软腐病(例如，禾本科软腐病，兰科软腐病；例如，玉米软腐病，兰花软腐病)；植物的茎基腐病(例如，玉米茎基腐病，番茄茎基腐病)；植物的灰霉病(例如，葡萄灰霉病)；
    优选地，所述逆境条件选自低温，干旱，高盐，药害(例如，化学药物)，土壤板结，酸性土壤，碱性土壤，高温环境，土壤肥力不足，或其任意组合；
    优选地，所述植物选自茄科，禾本科，豆科，葫芦科，十字花科，菊科，伞形花科，兰科；
    优选地，所述植物具有选自下列的一项或多项特征：
    (1)所述茄科植物选自番茄，辣椒，马铃薯，茄子，或其任意组合；
    (2)所述禾本科植物选自玉米，小麦，水稻，高粱，或其任意组合；
    (3)所述豆科植物选自大豆，花生，或其任意组合；
    (4)所述葫芦科植物选自黄瓜，冬瓜，南瓜，苦瓜，丝瓜，西瓜，罗汉果，或其任意组合；
    (5)所述十字花科植物选自白菜，油菜，甘蓝，萝卜，花椰菜，或其任意组合；
    (6)所述兰科植物选自兰花。
  18. 一种抑制植物的青枯病害的方法，该方法包括：将权利要求1所述的贝莱斯芽孢杆菌M173或其变体或其后代配制成生物农药制剂；将有效量的生物农药制剂施用于被青枯病原菌感染或有感染青枯病原菌风险的植物或其部分；优选地，所述植物是番茄或辣椒；优选地，述生物农药制剂通过喷雾、叶喷、浸根、灌根的方式施用。
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CN117757700A (zh) * 2024-02-04 2024-03-26 中国农业科学院生物技术研究所 一种贝莱斯芽孢杆菌及其应用
CN117757700B (zh) * 2024-02-04 2024-06-04 中国农业科学院生物技术研究所 一种贝莱斯芽孢杆菌及其应用
CN117701476B (zh) * 2024-02-05 2024-04-16 广东省科学院微生物研究所(广东省微生物分析检测中心) 一株对病原真菌具有拮抗作用的贝莱斯芽孢杆菌及其应用
CN117701476A (zh) * 2024-02-05 2024-03-15 广东省科学院微生物研究所(广东省微生物分析检测中心) 一株对病原真菌具有拮抗作用的贝莱斯芽孢杆菌及其应用
CN118006509A (zh) * 2024-04-03 2024-05-10 济南中科绿色生物工程有限公司 有抑菌促生作用的芽孢杆菌sgc3-2及其菌剂和应用

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