WO2017081105A1 - Souches de bacillus et agents ayant des propriétés bénéfiques - Google Patents

Souches de bacillus et agents ayant des propriétés bénéfiques Download PDF

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Publication number
WO2017081105A1
WO2017081105A1 PCT/EP2016/077179 EP2016077179W WO2017081105A1 WO 2017081105 A1 WO2017081105 A1 WO 2017081105A1 EP 2016077179 W EP2016077179 W EP 2016077179W WO 2017081105 A1 WO2017081105 A1 WO 2017081105A1
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Prior art keywords
bacillus subtilis
cell
agent
animal
plant
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PCT/EP2016/077179
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English (en)
Inventor
Zanmin Hu
Chengming FAN
Yuhong Chen
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Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences
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Priority to CN201680061786.5A priority Critical patent/CN108603161B/zh
Publication of WO2017081105A1 publication Critical patent/WO2017081105A1/fr

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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
    • C12N1/205—Bacterial isolates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Definitions

  • the present invention relates to agents comprising or derived from Bacillus species which have utility in for controlling plant diseases and promoting the health and growth of animals, including humans.
  • the invention further relates to materials and methods for identifying, preparing or using such agents.
  • Reducing the usage of artificial crop protection products and their residues, and plant preservatives, are important aims in improving the sustainability of the agricultural production system.
  • Novel agents are required for instance to overcome the problem of resistance to existing compounds, or to expand the range of diseases which can be controlled, or the ways in which existing agents are compounds. Novel agents having a biological origin may be preferable since they are typically less persistent in the environment.
  • the present inventors have identified a novel bacterial strain, identified herein as Bacillus subtilis HF1 , which shows utility as an agent for controlling plant diseases and also for promoting the health and growth of humans and other animals.
  • the invention relates, inter alia, to methods and materials for controlling plant diseases which are based on, or related to, Bacillus subtilis HF1 , or active compounds (for example fungicidal compounds) or mutants derived therefrom.
  • the invention further relates, inter alia, to methods and materials for controlling, inhibiting, or suppressing microorganisms, particularly fungi, which are based on, or related to, Bacillus subtilis HF1 , or active compounds (for example fungicidal compounds) or mutants derived therefrom.
  • microorganisms will preferably be pathogens e.g.
  • HF1 showed effect against a wide spectrum of fungal diseases, which suggests a utility in combatting fungal resistance. It is believed that HF1 can be used for controlling fungi spread by both soil and air.
  • Bacillus subtilis HF1 is not limited to the live strain.
  • the fermentation broth can be used either directly, dried, concentrated or in treated form, as well as a source of isolated or enriched active agents.
  • Bacillus subtilis HF1 can be used as a source of derived-strains, such as mutants having similar or improved properties.
  • HF1 products described herein which are based on, or related to, Bacillus subtilis HF1 , or active compounds or mutants derived therefrom, can be used as biofertilizers or biopesticides for combating plant diseases and improving plant health, or for preserving plant products, including derived food products, at or after harvest.
  • the present invention further relates to methods and materials for improving the growth or health of agricultural or other animals (including humans, poultry, fish and shellfish) using the HF1 products described herein.
  • agricultural or other animals including humans, poultry, fish and shellfish
  • they can be used as feed additives or veterinary or medicinal drugs for health improvement.
  • the invention provides bacterial isolates, probiotic formulations comprising the isolates and methods of using the probiotic formulations and isolates to improve the health or growth of animals.
  • the strain was identified as belonging to the species Bacillus subtilis based on the 16S rDNA analysis as set out in the Examples below.
  • This strain for instance in isolated or substantially isolated forms or cultures, form one aspect of the present invention.
  • the invention provides Bacillus subtilis HF1 strain having accession number CGMCC No. 1 1487 or a mutant thereof.
  • mutant thereof is meant a further strain derived from the deposited strain which has been altered in respect of one or more characteristics compared to the deposited strain. Methods of producing mutant strains are described below.
  • mutants will share one or more of the characteristics of deposited Bacillus subtilis HF1 strain described in Example 2 below, as well as the activities described herein.
  • Bacillus subtilis HF1 strain, or mutant thereof is in the form of a biologically pure culture.
  • Bacillus subtilis HF1 strain Bacillus subtilis is known for its ability to form small, tough, protective and metabolically dormant endospores. These spores of Bacillus subtilis HF1 strains, and their uses in the methods and processes described herein, form other aspect of the present invention.
  • the invention provides a composition comprising at least one Bacillus subtilis HF1 strain of the invention, in a suitable carrier.
  • suitable carrier are discussed in more detail hereinafter, but will be appropriate to the required utility e.g. controlling plant pathogens (an agriculturally acceptable carrier) or for improving animal health (e.g. a feed or additive to drinking water).
  • Endo a growth medium having the following composition is particularly suitable for growing the strains of the invention:
  • Mutants can be provided from the deposited Bacillus subtilis HF1 strain by those skilled in the art using standard methodologies.
  • UV ultraviolet light
  • NG N-methyl-N'-nitro-N-nitrosoguanidine
  • 4NQO 4-nitroquinoline-N-oxide
  • mutant strains of Bacillus subtilis HF1 strain capable of producing anti-microbial compounds or enhancing the growth of animals may be generated by UV mutagenesis.
  • mutants may be produced by exposure of the spores to UV light for a period of about 10 to about 180 seconds, preferably 45 to about 90 seconds, and most preferably 65 to 75 seconds.
  • Mutageneses involving NG may involve varying the concentration of the NG and exposure time.
  • NG at about 13 milligrams/liter (mg/L) to about 400mg/L of NG may be used for a period of exposure of about 15 minutes to about 120 minutes.
  • Alternative methods for generating mutants include techniques in the field of molecular biology.
  • oligonucleotide directed mutagenesis examples include, but are not limited to, oligonucleotide directed mutagenesis, linker scanning mutations or oligonucleotide directed mutagenesis using polymerase chain reaction (Ausubel (1987) Current Protocols in Molecular Biology).
  • selection criteria for screening mutants includes, but is not limited, to the ability of the bacillus colony to inhibit hyphal growth on a petri dish.
  • An non-limiting example of strain improvement via UV irradiation of a selected Bacillus subtilis strain to enhance production of antimicrobial metabolites is given by Krishna, Radha E., et al. "Strain Improvement of Selected Strain Bacillus subtilis (MTCC No. 10619) for Enhanced Production of Antimicrobial Metabolites.” RESEARCH JOURNAL OF BIOTECHNOLOGY 6.3 (201 1 ): 58-62.
  • aspects of the present invention include derived mutants of the deposited Bacillus subtilis HF1 strain, plus processes for preparing these by subjecting it to one or more rounds of mutagenesis, and culturing the resulting strains.
  • Bacillus subtilis HF1 cells may be prepared for use in the compositions using standard drying and fermentation techniques known in the art. Growth is commonly effected under aerobic conditions in a bioreactor at suitable temperatures and pH for growth. Typical growth temperatures are from 15 to 37°C, commonly 27°C to 32°C. Agitation may be used e.g. about 170 rpm.
  • Cultured material greater than or equal to about one, two, three or four days old may be used.
  • Typical concentration ranges for the Bacillus subtilis HF1 intact cells in the composition in the form of intact cells is from 1 x 10 3 to 1 x 10 14 , preferably 1 x 10 4 to 1 x 10 10 , more preferably 1 x 10 6 to 1 x 10 8 cells/mg. It will be appreciated that compositions with cell concentrates in order of 1 x 10 11 to 1 x 10 14 may be prepared and diluted before application if required.
  • the strains may be harvested using conventional washing, filtering or sedimentary techniques such as centrifugation, or may be harvested using a cyclone system.
  • Typical products may comprise cultured material of Bacillus subtilis HF1 without separation of spores and cells.
  • the practice of the invention is not limited to Bacillus subtilis HF1 strains, but also extends to products therefrom, either directly or via processing.
  • culture broth was used - for example after one or more of centrifugation (e.g. 10,000 rpm), heating (e.g. to 100°C for about 10 minutes), and also dilution (e.g. with water to between 0.1 % and 5% e.g. about 1 %, 0.5% or 0.25%).
  • centrifugation e.g. 10,000 rpm
  • heating e.g. to 100°C for about 10 minutes
  • dilution e.g. with water to between 0.1 % and 5% e.g. about 1 %, 0.5% or 0.25%.
  • Bacillus subtilis HF1 cells may be processed prior to use to produce active cell extracts, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates, cell pellets or may be used as whole cell preparations.
  • Other agents such as, but not limited to, Bacillus subtilis HF1 cells, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates, cell pellets or may be used as whole cell preparations.
  • Other agents may be used as whole cell preparations.
  • Such enrichment can be, for example, be based on bio-filtration using particularly diameter or molecular weight cut-off filters. For example in experiments below the supernatant was filtered using a biofilter having a diameter of 0.22 ⁇ .
  • the supernatant was sterilized at high temperature (121°C for 10 minutes).
  • methods of providing, or isolating, agents described herein may include heating to this temperature to confirm that the agent is substantially heat stable at this temperature.
  • substantially heat stable is meant that the agent retains the relevant activity when tested after heating the agent at the relevant temperature for 10 minutes, and preferably retains at least 50% of the untreated activity.
  • this and the other properties of the agents described herein can be utilised to purify them from, or enrich their concentration in, compositions derived from Bacillus subtilis strains and culture medium supernatants therefrom.
  • the activity assays described herein provide a convenient method of assaying the level of active agent after each stage, or in each sample of eluent.
  • one aspect of the invention provides a process for producing an isolated or encirched Bacillus subtilis HF1 agent, the process comprising isolating or enriching an active agent from a Bacillus subtilis HF1 source material, for example from a supernatant preparation, cell extract, cell suspension, cell homogenate, cell lysate, or cell pellet as described herein.
  • the process may comprise using one or more of the isolation techniques described above.
  • the activity of the agent can be assayed during the process e.g. for the ability to suppress the hyphal growth of a soil-borne phytopathogenic fungus or for promoting the growth or health of an animal.
  • Activity against pathogenic fungi can be assessed by ability of the agent to suppress the hyphal growth or fungal spore germination of a soil-borne filamentous phytopathogen fungus from one, two, three, four, five or preferably each of the following species:
  • the agents of the invention may lead to increase in production parameters for the treated animals as compared to controls. Production parameters include but are not limited to average daily weight gain, feed conversion rates, and consistency of growth among a group of animals.
  • Bacillus subtilis HF1 agent or similar will be understood to mean any agent based on or derived from the deposited Bacillus subtilis HF1 including, but not limited to: ⁇ The deposited Bacillus subtilis HF1 strain
  • compositions comprising one or more Bacillus subtilis HF1 agents as described above.
  • Such compositions preferably further comprise other materials or carriers appropriate to their purpose.
  • Bacillus subtilis HF1 agents for the control of plant disease or improving animal health
  • the invention provides a method for treating or controlling disease in a plant, the method comprising contacting the plant with a Bacillus subtilis HF1 agent of the invention.
  • Controlling in this context should not be taken to imply complete eradication of the disease or pathogen from the plant to be protected, and includes also suppressing or otherwise reducing or minimizing the effects of a pathogen, or increasing yields (or reducing death rates) in plants affected by the pathogen.
  • the plant is typically a crop plant.
  • Non-limiting Examples of plants which can be treated according to the invention are described in more detail below.
  • the disease is optionally a soil borne or air borne disease.
  • the disease is one caused by a phytopathogenic fungus.
  • a fungal target is powdery mildew.
  • the agent may be used to inhibit hyphal growth or fungal spore germination.
  • the invention provides a method for controlling at least one phytopathogenic fungus, the method comprising contacting the fungus with a Bacillus subtilis HF1 agent of the invention.
  • the method may be used to kill the pathogen.
  • the invention provides a method for soil remediation, the method comprising contacting the soil with a Bacillus subtilis HF1 agent of the invention.
  • the methods of the invention may be used to improve crop production.
  • the invention provides a method for preservation of an agricultural product (for example a harvested crop), the method comprising contacting the crop with a Bacillus subtilis HF1 agent of the invention.
  • Bacillus subtilis HF1 or mutants thereof are used in conjunction with other active agents e.g. pesticides or other disease control agents.
  • the invention provides a composition comprising at least one Bacillus subtilis HF1 agent of the invention, and an agriculturally acceptable carrier.
  • Bacillus subtilis HF1 agent(s) of the invention are present in the composition in an amount effective to control the disease or pathogen of interest.
  • the effective amount of control the disease or pathogen of interest is the amount effective to control the disease or pathogen of interest.
  • concentration may vary depending on the form the Bacillus subtilis HF1 agent is used in, the environment to which the composition is to be applied, the type, concentration and degree of disease or pathogen infestation; temperature; season; humidity; stage in plant growing season; age of plant; method, rate and frequency of application; number and type of conventional fungicides, pesticides and the like being applied, and plant treatments (for example pruning, grazing, and irrigation). All factors may be taken into account in formulating the composition.
  • compositions of the invention may be made by mixing one or more Bacillus subtilis HF1 agents with a desired agricultural carrier.
  • compositions of the invention may include humectants, spreaders, stickers, stabilisers, penetrants, emulsifiers, dispersants, surfactants, buffers, binders, and other components typically employed in known art insecticidal or control compositions.
  • composition of the invention may be in liquid or solid form, liquid compositions typically include water, saline or oils such as vegetable or mineral oils.
  • oils such as vegetable or mineral oils. Examples of vegetable oils useful in the invention are soy bean oil and coconut oil.
  • compositions may be in the form of sprays, suspensions, concentrates, foams, drenches, slurries, injectables, gels, dips, pastes and the like.
  • Liquid compositions may be prepared by mixing the liquid agriculturally acceptable carrier with the Bacillus subtilis HF1 agent. Conventional formulation techniques may be used to produce liquid compositions.
  • the compositions is in solid form.
  • the composition may be produced by drying the liquid composition of the invention.
  • a solid composition useful in the invention may be prepared by mixing Bacillus subtilis HF1 agents of the invention with a variety of inorganic or biological materials.
  • solid inorganic agricultural carriers may include carbonates, sulphates, phosphates or silicates, pumice, lime, bentonite, or mixtures thereof.
  • Solid biological materials may include powdered palm husks, corncob hulls, and nut shells.
  • the composition may be formulated as dusts, granules, seed coatings, wettable powders or the like.
  • the compositions may be formulated before application to provide liquid compositions.
  • compositions of the invention may be in the form of controlled release, or sustained release formulations.
  • Bacillus subtilis HF1 agent is applied to a plant or to agricultural produce.
  • Bacillus subtilis HF1 agent may be applied to the environment of the pathogen, typically around the plants to be protected, or the soil in which they have been or will be planted. Spraying, dusting, soil soaking, seed coating, foliar spraying, misting, aerosolizing and fumigation are all possible application techniques.
  • Application rates may for example be 10 10 spores/hectare to 10 14 spores, preferably 10 12 to 10 13 spores per hectare.
  • Typical application rates may be 50 g/hectare to 10,000 g/hectare. Commonly from 100 g/hectare to 5,000 g/hectare, or 500 to 1500 g/hectare.
  • the agents of the invention may be applied to seeds as a seed soak. This may be followed by drying.
  • the agents of the invention are applied to a plant as a seedling
  • Applications may be once only or repeated as required. Application at different times in plant life cycles, are also contemplated. For example, at or after harvest to prevent or minimize post-harvest disease.
  • a wide range of plants may be treated using the compositions of the invention.
  • Such plants include cereal, vegetable and arable crops, grasses, lawns, pastures, fruit trees and ornamental trees and plants.
  • Preferred plants for treatment are flowering plants and medical plants.
  • One example plant is Pseudostellaria heterophylla.
  • Arable crops which may particularly benefit from use of the compositions and strain(s) of the invention include crucifers and brassicas.
  • crucifers and brassicas For example, cabbage, broccoli, cauliflower, brussel sprouts and bok choy.
  • the invention provides a method for promoting the growth or health of an animal, the method comprising administering to the animal a Bacillus subtilis HF1 agent of the invention.
  • the methods typically include oral administration of the agent (e.g. as a "probiotic formulation" or animal feed) to an animal.
  • Oral administration includes, but is not limited to, delivery in feed (which includes in drinking water).
  • Alternatives include by oral gavage or aerosol spray.
  • the gastrointestinal health of the animal may be improved by the method. Promoting growth will typically lead to increased rate in a treated animal compared to a control animal not receiving the Bacillus subtilis HF1 agent of the invention - for example a weight increase of at least 5%, 10%, 15% or 20% - for example between 5 and 25%..
  • the animal is a poultry, more suitably a chicken or turkey.
  • the feed may comprise between 10 4 and 10 9 cfu bacteria/gm of finished feed.
  • the feed comprises between 10 5 and 5 x 10 7 cfu bacteria/gm feed.
  • the Bacillus subtilis HF1 agents may be added to the feed during production, after production by the supplier or by the person feeding the animals, just prior to providing the food to the animals.
  • the Bacillus subtilis HF1 strains used in the methods and compositions described herein are particularly suitable because they are capable of surviving (as spores) the heat and pressure conditions of the process of producing a dry pelleted feed product.
  • the animal is a human animal.
  • the animal is a farmed aquatic animal such as a fish, a crustaceans (e.g. shrimp, prawn, crab), or a mollusc.
  • a farmed aquatic animal such as a fish, a crustaceans (e.g. shrimp, prawn, crab), or a mollusc.
  • the invention provides an animal feed comprising a Bacillus subtilis HF1 agent disclosed herein.
  • the feed may (for example) comprise between about 10 4 and 10 9 cfu/gm finished feed.
  • the methods of increasing weight herein may be non-therapeutic.
  • the invention provides:
  • composition for the in the methods of treatment or therapy described herein.
  • Pulthogen refers to microorganisms (fungi or bacteria) that cause disease or damage to plants or animals.
  • the damage may relate to plant growth, yield,
  • pathogen is a filamental fungal organism that causes disease in plants.
  • the plants are cultivated plants.
  • an effective amount means an amount effective to control or eradicate pests, particularly insect pests.
  • biologically pure culture or “biologically pure isolate” as used herein refers to a culture of the relevant strain of the invention comprising at least 90%, preferably 95%, preferably 99% and more preferably at least 99.5% cells of the relevant strain.
  • animal includes mammals such as humans and farmed or companion or wild animals (e.g. a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape
  • Figure 1 The monoclonal morphology of HF1 cultivated on LB (Lysogeny Broth, 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCI) agar medium was irregular with lobate margins and folding surface.
  • FIG. 2 The growth curve of HF1 in different media. Note: LB, the LB liquid medium; 1 % and 2.8% Endo, the glucose concentration of medium was 1 % and 2.8% respectively.
  • Figure 3A Suppression effects of HF1 to important phytopathogens.
  • FIG. 5 The suppression of HF1 broth to the spore germination of Alternaria alternata. Note: A, HF1 broth was sterilized under 121 °C for 10 minuntes; B, HF1 broth was filtrated througth a biofilter. The broth was diluted by (A) 100 and (B) 500 x.
  • FIG. 7 Suppression effects of the HF1 LB broth on the powdery mildew.
  • the target crop of A and B was cucumber and pea respectively.
  • HF1 LB Broth contained living cells of HF1 were diluted by 300 x. Concentration of Azoxystrobin and ether phenolic were diluted by 1000 ⁇ according to the operating manual.
  • FIG 8 Bio-preservation effects of HF1 to strawberries. Note: A, Control; B, 0.2% Chitosan; C, HF1 with 0.2% Chitosan.
  • Figure 9 Growth promoting effects of HF1 on broiler chickens.
  • FIG 10 The phylogenetic tree of HF1 based on the 16S rDNAs. Note: the 16S rDNA sequnces from different species were downloaded from the NCBI database.
  • NR_024693.1 B.mycoides 273 , NR_036880.1 ; B.niacini IF015566, NR_024695.1 ;
  • B.anthracis Ames CP010792.1 ; B.cereus HN002, GQ478254.1 ; B.licheniformis ATCC 14580, CP000002.3; B.megaterium DSM 319, CP009920.1 ; B.pumilus SAFR-032, CP000813.1 ; B.subtilis 168, CP010052.1 ; B.thuringiensis 97-27, AE017355.1 .
  • Figure 1 1 - The relationship between the biomass (OD) and inhibition zone size
  • the HF1 was cultured in ENDO medium.
  • the OD was measured at different culture time and at same time the fungus growth inhibition was tested using the sterilized broth by cylinder plate method.
  • the fungus used is Alternaria alternata
  • the bacteria strain HF1 was isolated from the cucumber (Cucumis sativus) rhizosphere soil.
  • the contig15 (1627 bp) was the 16S rDNA sequence. This has the following sequence:
  • the 16S rDNA sequence of HF1 has the highest similar to that of Bacillus subtilis sp., for example Bacillus subtilis subsp. subtilis str. 168 (100%), which is the Bacillus subtilis standard stain. And then the phylogenetic tree was reconstructed through MAGE6.0 with Neighbor-joining method (see Figure 10) and show HF1 belonged to Bacillus subtilis spp.. At last, the strain HF1 was named as Bacillus subtilis HF1 , abbreviated to HF1 in the Examples hereafter.
  • BLAST Basic Local Alignment Search Tool
  • HF1 is a gram positive, aerobic, sporeforming bacteria, and its colony morphology cultivated on LB (Lysogeny Broth, 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCI) agar medium was irregular with lobate margins ( Figurel ).
  • HF1 is very sensitive to some antibiotics, such as Chloromycetin, Kanamycin,
  • Gentamicin Gentamicin, Spectinomycin, Rifampicin, Ampicillin, Tetracycline, Streptomycin, Penicillin and Carbenicillin.
  • the initial OD 6 oo value of HF1 was 0.267 ⁇ 0.0015 in 500 mL flasks with 200 mL LB liquid medium at 170 rpm and 28 °C.
  • the OD 6 oo of HF1 after inoculation for 24 hours reached 5.76 ⁇ 0.03 ( Figure 2).
  • HF1 An preferable medium for HF1 was found, and named "Endo", which contained glucose10-28 g/L, KN0 3 6 g/L, KH 2 P0 4 1.2 g/L, MgS0 4 -7H 2 01 .2 g/L, NasCeHsCv 0.2 g/L, CaCI 2 « 2H 2 0 105 mg/L, FeS0 4 « 7H 2 0 16 mg/L, EDTA 2.1 mg/L, H 3 B0 3 2.86 mg/L, ZnS0 4 « 7H 2 0 0.222 mg/L, MnCI 2 « 4H 2 0 1 .81 mg/L, Na 2 Mo0 4 0.021 mg/L and
  • Example 4 HF1 has a wide antimicrobial spectrum, showing HF1 is a great potential biocontrol agent to suppress plant diseases
  • Different fungous phytopathogens were separately inoculated on solid PDA medium (Potato Dextrose Agar, 200 g/L potato, 20 g/L dextrose, 10 g/L agar) and cultured at 28 °C for about 4 days.
  • solid PDA medium Potato Dextrose Agar, 200 g/L potato, 20 g/L dextrose, 10 g/L agar
  • Different agar disks with mycelium of 0.5 cm in diameters made by a punch from the margins of the colony with the same growth ability were inoculated on the middle of the Petri dish with PDA medium.
  • 1 ⁇ HF1 LB culture liquid was inoculated at about 2 cm away from agar disks. They were cultured for about 5 days and suppression effects of HF1 were investigated.
  • the strain HF1 can suppress the hyphal growth of some important soil-borne filamentous phytopathogen fungi ( Figure 3), such as Fusarium spp., Colletot chum spp., Rhizoctonia spp., Phytophthora parasitica., Alternaria spp.
  • Figure 3 some important soil-borne filamentous phytopathogen fungi
  • Gaeumannomyces graminis Verticillium dahliae, Calonectria nivale, Ceratobasidium cornigerum, Botryosphaeria berengeriana, Stemphylium solani, Coniothyrium spp, Phoma crystallifera, Myrmecridium schulzeri, Corynespora cassiicola and Magnaporthe grisea.
  • HF1 can be used as a biocontrol or soil remediation agent to suppress the soil borne disease.
  • Example 5 The broth of HF1 had inhibition activities to the hyphal growth
  • HF1 was cultured for about 2 days in LB at 170 rpm and 28 °C. After centrifugation, the supernatant was collected, and then a part of supernatant was sterilized under 121 °C for 10 minutes and the other was filtered by a biofilter (diameters 0.22 ⁇ ).
  • the fungus Alternaria alternata
  • PDA solid medium for about 5 days.
  • HF1 LB broth 1 ml_ of HF1 LB broth was prepared as in Example 5 in LB medium and added into 100 mL or 500 mL PDA medium. And then HF1 broth was diluted by 100 and 500 x, respectively. About 0.2 mL spore suspension liquid was spread onto the PDA solid medium plates.
  • Formulal Inhibitive rate (%) 2£rX 100% Note: CK, the number of colonies in controls; T, the number of colonies in treatments.
  • Example 7 HF1 can be used as a soil remediation agent for suppression of some soil- borne diseases and improving the crop production
  • Pseudostellaria heterophylla was selected.
  • the soil-borne diseases of P. heterophylla such as root rot caused by Fusarium oxysporum and Rhizoctonia solani and damping off caused by R. solani, can be very serious in the field because of the practice of successive planting.
  • a field with serious soil-borne diseases was used as the trial field.
  • the field was grouped into 10 trial blocks.
  • One group of 5 blocks was treated with the HF1 broth with living cells, and the others were used as the control. 300 seedlings were planted in every block (about 2.0 m long and 1.5 m wide).
  • the death rate (as the following Formula 2) was recorded at the harvest time.
  • the seeds were soaked by diluted 100 ⁇ HF1 broth with living cells for about 2 minutes and dried in the air in October. The seeds were then planted in the soil. After the emergence of seedlings, each of planting seedlings was watered with about 20 mL 300 ⁇ HF1 broth with living cells. When the next spring came, seedlings came back to growth, and were watered with about 20 mL 300 ⁇ HF1 broth with living cells for each plant. For the control, seedlings were watered with 20 mL water for each plant.
  • CK the number of plants in controls
  • T the number of plants in treatments
  • HF1 can be a potential soil remediation suppressing the soil-borne diseases and improving the crop production.
  • HF1 was cultured in LB liquid medium.
  • the broth with living cells was called "HF1 LB broth”.
  • HF1 LB broth with living cells was diluted by 300x and applied as a foliar spray
  • the disease grading as the following: 0, no spots; 1 , the ratio of the infected area and the whole leaf area was less than 25%; 3, the rate of the infected area and the whole leaf area was between 25% and 50%; 5, the rate of the infected area and the whole leaf area was between 50% and 75%;7, the rate of the infected area and the whole leaf area was more than 75% 0
  • the disease indexes (%) were ⁇ (the disease gradingxnumber of the corresponding leaves)/(The total number of the investigated leavesxthe highest disease grading)]x100%.
  • Control effects (%) were [1 - (disease index of the treatment/disease index of the control)] *100%.
  • Example 9 HF1 LB broth can be used as a bio-preservative
  • the HF1 broth was heated at 100 °C for about 10 minutes and centrifuged at 12,000 rpm. The supernatant was collected and diluted by 100 x. Perishable strawberry was the target fruit. The strawberries were soaked in sterile water (the negative control) and 0.2% chitosan or 100 ⁇ supernatant of HF1 LB broth with 0.2% chitosan for about 20-30 seconds and air dried. The treated fruit was put into fresh-keeping boxes at room temperature. Ten strawberries were used for each treatment and every treatment was replicated for three times.
  • HF1 LB broth can be used as a bio-preservative.
  • Example 10 HF1 broth can be used as the feed additive to promote the growth of target animals.
  • the low doses of antibiotics can promote the animal body weight, but can seriously affect the animal, and ultimately human, health, and also damage the environment.
  • Bacillus subtilis had been approved as an additive by the ministry of agriculture for use in feeding white feather broiler. Compared with negative controls, the final weight increase is about 3.5% by using regular Bacillus subtilis strain, which is not significantly different to antibiotic feeding.
  • HF1 was cultured in liquid LB and Endo medium, respectively.
  • the broth was heated at 100°C for about 10 minutes after 10,000rpm centrifugation.
  • the LB broth was diluted by water to 1 %, 0.5% and 0.25%, and the Endo broth was also diluted by water to 1 %.
  • the diluted broth was used as the daily drinking water for the broiler chicken (Gallus gallus domesticus Baiyu) fed on basal feed from 1 day to 42 days.
  • the broiler chickens fed on the basal feed and water without any additive were used as the negative control (CK); and those fed on the basal feed with 15 mg/kg virginiamycin as additive were used as the positive control.
  • the average weight of chickens drinking 1 %, 0.5% and 0.25% LB broth and 1 % Endo broth was 2771.90 ⁇ 19.06 g, 2698.38 ⁇ 13.92 g, 2651.55 ⁇ 6.28 g, 2775.23 ⁇ 39.69 g respectively when trials were finished on day 42.
  • the average weight of CK and the positive control were 2309.90 ⁇ 26.39 g and
  • the weight increasing ratio of chickens drinking 1 %, 0.5% and 0.25% LB broth and 1 % Endo broth were 20.01 ⁇ 1.85%, 16.82 ⁇ 1 .79%, 14.80 ⁇ 1.73%
  • the weight increasing ratio of chickens drinking 1 %, 0.5% and 0.25% LB broth and 1 % Endo broth were 13.73 ⁇ 1.33%, 10.71 ⁇ 1.24%, 8.79 ⁇ 1.16%, 13.86 ⁇ 1.26%.
  • HF1 group weight is significant higher than those of the negative control and positive controls: compared with negative control, the weight was increased by 28%; compared with a commercial Bacillus subtilis probiotic supplement and different antibiotics (virginiamycin, amoxicillin), the weight was increased by 16% and 25%. Additionally the beneficial activity was demonstrated when chickens were fed with HF1 culture dried on a solid, either starch or a rice husk / starch mixture (results not shown).
  • Example 1 The stain HF1 was safe to animals tested by the acute toxicity trial.
  • CD-1 mice with 20 ⁇ 2 g were selected as the target animals to investigate the acute toxicity of HF1.
  • the mice were divided into 5 groups. Each group had six mice with 3 males and 3 females. The mice were not fed one day before the trial. Each group was separately given with 0.4 ml 0.8% NaCI, 10 9 cfu/mL, 10 11 cfu/mL, 10 12 cfu/mL and HF1 LB broth without dilution, once by gavage. And then mice were reared normally for about 15 days. All the mice were killed 15 days later. The clinic performances of the mice were observed.
  • mice All tested mice were alive at the end of experiments.
  • the clinic performances of the tested mice were not different from those of the control (the group 0.8% NaCI).
  • the clinic performances include daily feeding, drinking, coat color, faeces, weight, death rate, and general anatomical results. According to the anatomical results, there were no visually abnormal tissues in any of the mice. Acute toxicity tests showed that Bacillus strain HF1 should be safe to mammals.

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Abstract

L'invention concerne une nouvelle souche bactérienne, identifiée comme Bacillus subtilis HF1, qui présente une utilité comme agent pour lutter contre des maladies des plantes et également pour favoriser la croissance d'animaux. L'invention concerne des agents, des procédés, des traitements, des utilisations et des compositions ayant une utilité pour ces objets, comportant la souche, ou des dérivés de ces derniers.
PCT/EP2016/077179 2015-11-09 2016-11-09 Souches de bacillus et agents ayant des propriétés bénéfiques WO2017081105A1 (fr)

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WO2019040266A1 (fr) * 2017-08-23 2019-02-28 Novozymes A/S Agents microbiens alimentés directement pour améliorer l'état général et la santé des poissons
JP2019054786A (ja) * 2017-09-20 2019-04-11 行政院農業委員會農業薬物毒物試驗所 細菌発酵羽毛粉末を調製するためのバチルス・サブティリス株及びその使用
WO2019152791A1 (fr) * 2018-02-02 2019-08-08 Novozymes A/S Prise en charge du pathogène lawsonia
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