CN108603161A - Bacillus strain with beneficial activity and preparation - Google Patents
Bacillus strain with beneficial activity and preparation Download PDFInfo
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- CN108603161A CN108603161A CN201680061786.5A CN201680061786A CN108603161A CN 108603161 A CN108603161 A CN 108603161A CN 201680061786 A CN201680061786 A CN 201680061786A CN 108603161 A CN108603161 A CN 108603161A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
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- 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
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Abstract
The present invention provides a kind of new bacterium bacterial strains, are identified as bacillus subtilis HF1.The bacterial strain can be used as preparation, and said preparation is for controlling plant disease, it can also be used to improve growth of animal.The present invention provides preparation, method, purposes and the composition in terms of can be used for these according to the bacterial strain or derivatives thereof.
Description
Technical field
The invention mainly relates to the preparation comprising or derived from bacillus, said preparation can be used for control plant disease and
Improve growth and the health of animal (including mankind).
The invention further relates to identification, preparation or use the material and method of this preparation.
Background technology
The use for reducing artificial crop protection products and its residue and plant antistaling agent, for realizing agricultural production system
The sustainable development important in inhibiting of system.
Therefore, people are for having the ability preventing plant disease preparation and material of disease as caused by Filamentous disease fungus
Demand is growing day by day.Wish that novel formulation can solve the problems, such as the resistance for such as overcoming disease to existing pesticide, or can expand to disease
Preventive effect range or the method that does not use of existing chemical pesticide.The preparation of biological source in the environment will not be forever due to it
It remains and gains great popularity long.
Furthermore application of the antibiotic in terms of maintaining animals and humans health is especially supported in poultry, fish and shellfish etc.
Application in growing is by the pressure more and more from government administration section and consumer.This causes the efficient replacement of antibiotic
Product become a kind of demand.The use of directly edible microorganism or its active material generated will be a kind of potential alternative solution.
Invention content
Inventor identifies a kind of new bacterium bacterial strain, and is named as Bacillus subtillis HF1 (Bacillus
subtilis HF1).The bacterial strain can be used as preparation, and said preparation is dynamic with other for controlling plant disease and being additionally operable to improvement people
The health of object and growth.
The invention particularly relates to the method and material for controlling plant disease, the method and material are based on or are related to withered
Careless bacillus HF1 or by its derivative active material (such as Fungicidal compounds) or mutant strain.
The present invention also relates particularly to the method and material for controlling, inhibiting microorganism especially fungi, the method and
Material is based on or is related to bacillus subtilis HF1 or by its derivative active material (such as Fungicidal compounds) or its mutant strain.
Microorganism is preferably pathogen, such as phytopathogen.
In embodiment thereafter, HF1 strains expresseds go out the effect of the prevention fungal disease of wide spectrum, this shows that it can be used for
Prevent Antifungal resistance.Now think that HF1 can be used for controlling the fungi propagated by both soil and air.
In addition, what following article was confirmed, the application of bacillus subtilis HF1 bacterial strains is not limited only to its viable bacteria.Its zymotic fluid
Also can directly apply, or by drying, concentration or in other ways processing formed product by apply, from the bacterium separation or it is dense
The active ingredient of contracting can also be applied.
In other respects, bacillus subtilis HF1 can be used as the source of derivative strain, such as with spy that is similar or improving
The mutant strain of property.
The product of HF1 described herein is based on or is related to HF1 bacterial strains or by its derivative active material or mutant strain, the production
Product can be used as preventing plant disease and improve plant health, or when for making harvest or after harvest plant product (including
Derivative food) fresh-keeping bio-feritlizer or biological insecticides.
The invention further relates to use HF1 products described herein improve agricultural animal or other animals (including people, poultry,
Fish and shellfish) growth and health method and material.It can such as improve as feed addictive or veterinary drug or medical drugs
Health.In certain embodiments, the probiotic formulations object the present invention provides bacterium isolated strains, comprising the isolated strains
And using the probiotic formulations object and isolated strains to improve the method for the health and growth of animal.
We will illustrate in further detail below in terms of these.
The bacterial strain and mutant strain of preservation
For proving that the specific bacterial strain bacillus subtilis HF1 of the preparation of the present invention has been deposited in the micro- life of the Chinese Academy of Sciences
The China General Microbiological culture presevation administrative center (CGMCC) (according to budapest treaty) of object research institute, address are north
The accession number of the institute of the Chaoyang Districts Jing Shi North Star West Road 1, postcode 100101, the bacterial strain is CGMCC No.11487, preservation date
For on October 13rd, 2015.
The 16S rDNA analyses listed according to Examples below, the bacterial strain are accredited as belonging to bacillus subtilis.
Embodiment 2 illustrates some characteristics of the bacterial strain.
The bacterial strain (such as the form of separation or form or culture for being basically separated) constitutes the part of the present invention.
On the other hand, the present invention provides accession number be CGMCC No.11487 bacillus subtilis HF1 bacterial strains or its
Mutant strain.
" its mutant strain " refers to the bacterial strain derived by preservation strain, these bacterial strains are compared with preservation strain, at one
Or there is change in multiple characteristics.The preparation method of mutant strain will be described below.
Typically, there are one preservation strain bacillus subtilis HF1 is total to during these mutant strains are described with embodiment 2 below
Or multiple features and activity described herein.
In one embodiment, bacillus subtilis HF1 bacterial strains or by its mutant strain with culture pure on biology
Form exists.
Unless the context specifically indicates, if herein use " bacillus subtilis HF1 bacterial strains ", will all refer to preservation strain and
By its derivative mutant strain.
It is well known that bacillus subtilis can generate the endospore of small, tough and tensile, protective metabolism suspend mode.Cause
This, these spores of bacillus subtilis HF1 and its in method described herein and in the process application constitute the present invention its
In terms of him.
In one aspect, the present invention provides the bacillus subtilis for being included at least one of suitable carrier present invention
The composition of HF1.This carrier is described in greater detail below, but these carriers are suitable for required application
, required application is as controlling phytopathogen (agriculturally acceptable carrier) or for improving animal health (as made
For feed or the additive being added in drinking water).
The growth and culture of Bacillus strain
Inventor provides a kind of growth medium (being named as herein " Endo "), its composition is as follows：Glucose 10-
28g/L, KNO36g/L, KH2PO41.2g/L, MgSO4·7H2O 1.2g/L, Na3C6H5O70.2g/L, CaCl2·2H2O
105mg/L, FeSO4·7H2O 16mg/L, EDTA 2.1mg/L, H3BO32.86mg/L ZnSO4·7H2O 0.222mg/L,
MnCl2·4H2O 1.81mg/L, Na2MoO40.021mg/L and CuSO4·5H2O 0.07mg/L。
It grows, cultivate using the culture medium or ferment Bacillus strain as the bacterial strain of the present invention constitutes the present invention
On the other hand.
The mutant strain of preservation strain
Mutant strain can be obtained using standard method from bacillus subtilis HF1 by those skilled in the art.
For example, it will be understood by those skilled in the art that the method for DNA mutagenesis or mutant strain screening is well known, including a variety of
Chemistry or enzyme method.The example of these methods includes but not limited to be exposed to ultraviolet light (UV), nitrous acid, N- methyl-N '-nitre
Base-N- nitrosoguanidines (NG), 4- nitroquinoline-N- nitrogen oxides (4NQO).(Leninger(1972)
Biochemistry.Worth Publishers Inc.,NY；Jeffrey H.Miller(1972)"Experiments in
Molecular Genetics.Cold Spring Harbor Laboratory,Cold Springs Harbor,New
York).Preferred method includes UV and NG.
For example, the mutant strain of bacillus subtilis HF1 bacterial strains, the mutation can be generated by ultraviolet radioactive mutagenesis
Bacterial strain can generate antibacterial material or enhancing growth of animal.For example, by the way that spore is exposed about 10 to about 180 seconds under ultraviolet light
Mutant strain can be obtained in (preferably 45 seconds to about 90 seconds, most preferably 65-75 seconds).Be related to NG mutagenesis may include change NG concentration and
Exposure duration.By taking NG as an example, a concentration of about 13mg/L to about 400mg/L NG can be used to expose about 15 minutes to about 120 minutes
Time.The alternative for obtaining mutant strain includes the technology of molecular biology field.The example of these technologies includes but not limited to
Oligonucleotides directed mutagenesis, linker scanning mutagenesis (linker scanning mutation) and using PCR
Oligonucleotides directed mutagenesis (Ausubel (1987) Current Protocols in Molecular Biology).
Screening and selection can pass through biography with improvement or the mutant strain of the production of improved antiseptic or enhancing growth of animal
Those of the assay method of system carries out, such as be described herein.The example of screening criteria for screening mutant strain includes but unlimited
In the ability that bacillus clone inhibition mycelia grows in petri dish.Krishna, Radha E et al., " Strain
Improvement of Selected Strain Bacillus subtilis(MTCC No.10619)for Enhanced
Production of Antimicrobial Metabolites.”RESEARCH JOURNAL OF BIOTECHNOLOGY6.3
(2011):58-62 gives following non-limiting examples：By carrying out ultraviolet radioactive to selected bacillus subtilis strain
The bacterial strain improvement of antibacterial metabolin generation can be enhanced by obtaining.
Therefore, many aspects of the invention include the derivative mutant strain of bacillus subtilis HF1 bacterial strains and by it
Carry out a wheel or the method for taking turns mutagenic treatment and preparing these mutant strains and cultivating obtained strains more.
Composition and Related product
It can be dried by standard and zymotechnique is obtained for the bacillus subtilis HF1 cells in composition.Growth is logical
Often carry out in the bioreactor, under aerobic conditions, under the conditions of the temperature and pH suitable for growth.Typically growth temperature is
15-37 DEG C, usually 27 DEG C -32 DEG C.Rotating speed is for example, about 170rpm.
Culture culture of the age greater than or equal to about 1 day, 2 days, 3 days or 4 days can be used.
It is general a concentration of the 1 × 10 of the bacillus subtilis HF1 intact cells of intact cell form in composition3-1×
1014A cell/mg, preferably 1 × 104-1×1010A cell/mg, more preferable 1 × 106-1×108A cell/mg.It should be understood that
It is 1 × 10 that cell concentration, which can be prepared,11-1×1014The composition of a cell/mg, if necessary can be dilute by its before administration
It releases.
Bacterial strain can either sedimentation collects or uses cyclone system (as centrifuged) by traditional washing, filtering
It collects.The cell of collection can immediately using or be deposited under cold as at 4 DEG C.Preferably after cell collection as early as possible
It uses.
In other embodiments, cell or zymotic fluid product (with and without cell) can be frozen using conventional method
It is dry or dry, such as be spray-dried under heating or vacuum condition.Preferred method is freeze-drying.Suitable for bacillus
Freeze Drying Technique non-limiting examples it is on the books in 2008041786 A1 of WO.
Typical product may include the culture of bacillus subtilis HF1, not detach spore and cell.
The practice of the present invention is not limited to bacillus subtilis HF1 bacterial strains, no matter also extends into the product formed by it
It is directly formed or is formed by processing.
For example, in the following embodiments, using cultivation and fermentation liquid-for example one or many centrifugations (such as 10,
000rpm), it heats (such as be heated to 100 DEG C and last about 10 minutes) and is also diluted and (such as be diluted with water to a concentration of
0.1%-5%, than such as from about 1%, 0.5% or 0.25%) after zymotic fluid.
Therefore in terms of different, bacillus subtilis HF1 cells can be processed before use to generate competent cell
Extract, cell suspension, cell homogenates, cell pyrolysis liquid, cell supernatant, cell filtrate, cell mass, or may be used as complete
Cell preparation.
Other preparations
If necessary, it can be purified from the cell and culture of bacillus subtilis HF1 or its mutant strain or rich
Collection (as used methodology discussed below) bacillus subtilis HF1 preparations.
This enrichment can be based on the biofiltration for using special diameter or weight shutoff filter.Such as following
In experiment, the biofilter of 0.22 μm of diameter of supernatant filters.
In other embodiments, supernatant is by high-temperature sterilization (121 DEG C sterilize 10 minutes).Therefore it provides or detaches this
The method of the preparation of text description may include preparation being heated to the temperature to confirm that said preparation is substantially hot steady at such a temperature
Fixed.
" substantially heat-staple " refers to the system when being tested preparation heating after ten minutes under associated temperature
Agent retains related activity, preferably retains untreated active at least 50%.
If necessary, using the characteristic of preparation described herein and other characteristics with from derived from withered grass bud
The concentration of purifying said preparation or increase said preparation in the composition of spore bacillus strain and the medium supernatant being generated by it.
Activity test as described herein provides a kind of method of simplicity, and this method measures after each stage or each elution
The level of active material in liquid sample.
From the method for purified peptide in heterogeneous mixture or other compounds in the art in be that comparison is clear,
For example selective precipitation, proteolysis, with known molecular amount cut-off filter carry out ultrafiltration, ion-exchange chromatography, gel mistake
Filter etc..One particularly useful technology of this respect is supercentrifugation (for example 150,000 × g centrifugations was more than 1 hour).Other
The known method suitable for protein purification has disclosure in the following：“Methods in Enzymology Vol 182-Guide
to Protein Purification”Ed.M P Deutscher,Pub.Academic Press Inc.Typical method is also
In " Protein Purification-principles and practice " Pub.Springer-Verlag, New York
Inc (1982) and Harris＆Angal (1989) " Protein purification methods-a practical
It is listed in approach " Pub.O.U.P.10UK or bibliography or its later release.
Therefore, the method that one aspect of the invention provides the bacillus subtilis HF1 preparations for generating separation or enrichment, it is described
Method includes separation or deposition activity substance from bacillus subtilis HF1 raw material, is such as prepared from supernatant described herein
In object, cell extract, cell suspension, cell homogenates, cell pyrolysis liquid or cell mass.This method may include using above-mentioned
One or more isolation technics.In this approach, the activity of preparation can be measured, such as preparation inhibits soil biography pathogenic true
The mycelia of bacterium grows or improves the ability of growth of animal or health.
Disease-resistant fungal pathogen activity can inhibit mycelia growth or the allergenic of soil biography nematophyte disease fungus by preparation
The ability that son is sprouted is assessed, and the soil passes nematophyte disease fungus and comes from a kind below, 2 kinds, 3 kinds, 4 kinds, 5 kinds or excellent
Be selected as it is below each：Fusarium spp.、Colletotrichum spp.、Rhizoctonia spp.、
Phytophthora spp.、Alternaria spp.Gaeumannomyces graminis、Verticillium
dahliae、Calonectria nivale、Ceratobasidium cornigerum、Botryosphaeria
berengeriana、Corynespora cassiicola。
The preparation of the present invention can be such that processed animal is improved in terms of manufacturing parameter compared with the control.Manufacturing parameter packet
The growth consistency for including but being not limited in averagely every daily gain, feed conversion rate and animal groups.
According to the above it is appreciated that term as used herein " bacillus subtilis HF1 preparations " or similar word can
To be understood as referring to any preparation of the bacillus subtilis HF1 based on or derived from institute's preservation, including but not limited to：
● the bacillus subtilis HF1 bacterial strains of preservation；
● the derivative mutant strain generated by the bacterial strain；
● the spore of bacillus subtilis HF1 bacterial strains；
● the supernatant from bacillus subtilis HF1；
● from bacillus subtilis HF1, partial purification or the supernatant of concentration；
● it is originated from any active material in these, the substance one or more activity described herein, such as in soil
It passes the activity in terms of nematophyte disease fungus or the activity of broiler weight or animal health can be improved.
Just as set forth above, the present invention provides including one or more bacillus subtilis HF1 as described above
The composition of preparation.This composition preferably also contains the other materials or carrier of the purposes suitable for the composition.
Other aspects of the present invention include using bacillus subtilis HF preparations control plant disease or improving animal health
Method.
The preferred method of the present invention
Therefore, on the one hand, the present invention provides a kind of method for treating or controlling plant disease, the method includes
Plant and the bacillus subtilis HF1 preparations of the present invention is set to contact.
" control " mentioned in context should not be regarded as it means that eliminated completely from plant to be protected disease or
Pathogen further includes inhibiting or otherwise reducing or minimize the influence of pathogen, or increase and catch pathogen
The yield (or reducing the death rate) of plant.
The plant is typically a kind of crops.It can be according to the non-limiting examples of plant that the present invention is handled hereinafter
It is described in more detail.
The disease is optionally a kind of soil or airborne disease.
Preferably, the disease is the disease caused by plant pathogenic fungi.The example of fungal disease is powdery mildew.
This preparation can be used for inhibiting mycelia growth or fungus spore germination.
Therefore, the present invention provides the method for controlling at least one plant pathogenic fungi, this method includes making the present invention's
Bacillus subtilis HF1 preparations and Fungal contact.This method can be applied to kill pathogen.
The present invention provides a kind of method for soil remediation, this method includes making the bacillus subtilis of the present invention
HF1 preparations contact soil.
The method of the present invention can be used for improving crop yield.
The present invention provides a kind of method fresh-keeping for agricultural product (such as crop of harvest), this method includes making this hair
It is bright that bacillus subtilis HF1 preparations contact crop.
Optionally, it derives from bacillus subtilis HF1 or the substance of its mutant strain can be with other agents such as insecticide
Or other diseases controlling agent is used in combination.
For plant
On the other hand, the present invention provides a kind of composition, and the composition contains the bacillus subtilis of at least one present invention
Bacterium HF1 preparations and agriculturally acceptable carrier.
The bacillus subtilis HF1 preparations of the present invention are present in the combination with the amount of effective control targe disease or pathogen
In object.Effective concentration depends on：The use form of the bacillus subtilis HF1 preparations；The environment of the composition to be administered；Disease
Or type, concentration and degree, the temperature of pathogenic bacterial infection；Season；Humidity；Stage of the plant in the season of growth；Plant age；It applies
Method, amount of application and frequency；The quantity and type of the conventional fungicides and agrochemical applied；The processing of plant
(such as trim, herd and irrigate).When preparing the composition, all of these factors taken together may all be taken into account.
The composition of the present invention can be by by one or more bacillus subtilis HF1 preparations and required agricultural acceptable carrier
It mixes and is made.
The present invention composition may include moisturizer, spreading agent, paste agent, stabilizer, bleeding agent, emulsifier, dispersant,
Surfactant, buffer, adhesive and other compositions commonly employed in the art, insecticide or control composition.
The composition of the present invention can be liquid or solid form, and liquid composition generally includes water, brine or oils (such as
Vegetable oil or mineral oil).The example of vegetable oil used in the present invention is soya-bean oil and coconut oil.
The composition can be following form：Spraying, suspension, concentrate, foam, immersion liquid, slurries, injectable agent, gel,
Dip, paste etc..
Liquid composition can be prepared by the agriculturally acceptable carrier of mixes liquid with bacillus subtilis HF1 preparations
It forms.Traditional formula technique can be used for producing liquid composition.
In one embodiment, the composition is solid form.The composition can be passed through by the liquid composition of the present invention
Drying is made.In addition, solid composite for use in the present invention can be by by the bacillus subtilis HF1 preparations of the present invention and respectively
The inorganic or biomaterial of kind is mixed with.For example, solid inorganic agricultural acceptable carrier may include carbonate, sulfate, phosphoric acid
Salt or silicate, float stone, lime, bentonite or their mixture.Solid bio-material may include powdered palm shell, corn
Crust and peanut shell etc..
The composition can be configured to pulvis, granule, seed pelleting, wettable pulvis etc..It can prepare before administration
The composition is to provide liquid composition.
The composition of the present invention can be the form of controlled or sustained release formulations.
In one embodiment, bacillus subtilis HF1 preparations are administered to plant or agricultural product.
As described above, bacillus subtilis HF1 preparations can be applied to the environment residing for pathogen, usually plant to be protected
Around object or plants and be implanted with plant or the soil by planting plants.Spraying, dusting, soil immersion, seed pelleting, blade face spray
It is all the method that possible use to spill, be atomized and fumigate.
For example, amount of application can be 1010-1014A spore/hectare, preferably 1012-1013A spore/hectare.
Common amount of application can be 50-10000 grams/ha.Generally 100-5000 grams/ha or 500-1500 grams/
Hectare.
In addition, the preparation of the present invention can be applied to seed in such a way that seed impregnates, it then can be again by seed drying.
In addition, the preparation of the present invention is also applied to plant seedlings.
It according to demand can primary or repetitive administration.The different time being also contemplated in plant life cycle is administered.
For example, being applied in harvest or after harvest, to prevent or reduce the disease after harvest.
Various plants can be handled using the composition of the present invention.This plant include cereal, vegetables and cultivable crop,
Grass, lawn, pasture, fruit tree and ornamental trees and plant.Preferred plant is flowering plant and medicinal plant.
Such as radix pseudostellariae (Pseudostellaria heterophylla) this plant.
May include crucifer in particular benefit from the composition of the present invention and the cultivable crop of bacterial strain used
And Brassica plants.Such as cabbage, broccoli, cauliflower, brussels sprout and Chinese cabbage.
For animal
Therefore, on the one hand, the present invention provides it is a kind of improve animal growth and health method, the method includes to
The bacillus subtilis HF1 preparations of the animal application present invention.
This method generally includes said preparation (such as " probiotics preparation " or animal feed) being administered orally to animal.
It includes but not limited to delivering in feed (including drinking water) to be administered orally, and alternative solution further includes molten by oral administration gavage or gas
Glue spraying application.
In this way, the gastrointestinal health situation of animal can be improved.
Compared with control-animal (the bacillus subtilis HF1 preparations for not receiving the present invention), improving growth would generally carry
Growth rate --- weight of for example, at least 5%, 10%, 15% or 20% increases --- such as 5- of high processed animal
25%.
Suitable animal is poultry, more preferably chicken or turkey.If supplied in animal feed, every gram of finished product of feed
Feed may include 104-109Cfu bacteriums.Suitable every gram of feed of feed includes 105-5×107Cfu bacteriums.Bacillus subtilis
HF1 preparations can during production or production after be added in feed by supplier, or just to animal provide food it
The preceding personnel by raising animal are added in feed.Bacillus subtilis HF1 bacterium for method described herein and composition
Strain is particularly suitable, because they can be with spore under the heating of dry particle feed product production method and pressure condition
Mode survive.
In other embodiments, animal is people.
Providing improves the method for the gastrointestinal health in this animal.
In other embodiments, animal is the aquatic animal of cultivation, such as fish, Crustaceans (such as shrimp, prawn, crab)
Or mollusk.
The method for additionally providing prevention animal fungal infections.
Therefore, on the one hand, the present invention provides a kind of animal feeding containing bacillus subtilis HF1 preparations disclosed herein
Material.The wherein described preparation is bacterial strain or spore, and every gram of finished product feedstuff of the feed can include about 104-109Cfu bacteriums
Weight increasing method herein can be therapeutic with right and wrong.
However, in other respects, the present invention provides：
(i) bacillus subtilis HF1 preparations disclosed herein are in therapy described herein or therapy；
(ii) bacillus subtilis HF1 preparations disclosed herein are being prepared in therapy described herein or therapy
Composition (drug and veterinary products) in application.
Definition
" pathogen " refers to causing plant or animal diseases or (fungi is thin to plant or the hurtful microorganism of animal
Bacterium).For example, this damage can be related with the growth of plant, yield, breeding or survival ability, can also be the damage on surface.It is excellent
Selection of land, damage refer to the damage for having Economic Importance.In preferred embodiments, term " pathogen " is to cause phytopathy
Harmful filamentous fungi.Plant is preferably cultivated plant.
"comprising" this term is used in specification and means " at least by ... form ".Include art when illustrating book
When each of language "comprising" is stated, other than starting those features with term, other features also may be present.Some related arts
Language, such as term "comprising" and " containing " explain in the same way.
In the description term " substantially by ... form " refer to that described feature and allowing occurs will not be real
Change to matter other features of the fundamental characteristics of described characteristic.
Term " effective quantity " effectively controls or eliminates pest, especially insect pest with herein referring to one,
Amount.
Term " pure culture on biology " or " pure separation strains on biology " are with referring herein to related strain of the present invention
Culture, it includes the cells of at least 90%, preferably 95%, preferably 99%, more preferably at least 99.5% related strain.
" animal " this term includes the mammal such as mankind and livestock either companion animals or wild animal (example
Such as, placental mammals, marsupial (such as kangaroo, wombat), monotreme (such as platypus), rodent (example
Such as cavy, hamster, rat, mouse), murine (such as mouse), rabbit section animal (such as rabbit), canid (such as dog),
Felid (such as cat), equid (such as horse), porcine animals (such as pig), caprid (such as sheep), bovid
(such as ox), primate, anthropoid (for example, monkey or ape), monkey (such as marmoset, baboon), ape (such as gorilla, chimpanzee,
Orangutan, gibbon), also fish, birds, reptile, Crustaceans, mollusk.Preferably, farm's cultivated animals
And aquiculture animal.
Description of the drawings
Fig. 1-culture LB (peptone of Lysogeny Broth, 10g/ml, the yeast extract of 5g/L, 10g/L's
Nacl) colonial morphology of the HF1 on agar medium is irregular, the surface with lobate edge and folding.
The growth curves of Fig. 2-HF1 in different medium.Annotation：LB, LB liquid medium；1% and 2.8%Endo,
The concentration of glucose is 1% and 2.8% respectively in culture medium.
Depression effects of Fig. 3 A-HF1 to important phytopathogen.
Inhibitions of Fig. 3 B-HF1 to fungi：A=rice blast fungus (rice blast strain) bacterial strain 1-2-5；B=rice
Pest bacterium (Magnaporthe grisea) appresorium bacterial strain 3-2；C=rice blast bacteria strains 5-2-2.
The HF1 zymotic fluids of the not no living cells of Fig. 4-are to glue born of the same parents anthrax-bacilus (Colletotrichum gloeosporiodes)
Mycelia growth inhibition.Annotation：A, HF1 zymotic fluid sterilize 10 minutes at 121 DEG C；B, HF1 zymotic fluid are filtered by filter.
The inhibition of the spore germination of Fig. 5-HF1 fermentation broth on tobacco rust opportunistic pathogen (Alternaria alternata)
Effect.Annotation：HF1 zymotic fluids sterilize 10 minutes at 121 DEG C；B, HF1 zymotic fluid are filtered by biofilter.(A) zymotic fluid
100 times of dilution, (B) zymotic fluid dilute 500 times.
Biological control effects and improvement radix pseudostellariae (Pseudostellaria of Fig. 6-HF1 to soil-transmitted disease
Heterophylla) the effect grown.
Depression effect of Fig. 7-HF1LB zymotic fluids to powdery mildew.Annotation：The target crop of A and B is cucumber and pea respectively
Beans.HF1LB zymotic fluids include the living cells of HF1, and are diluted 300 times.It is according to operational manual, Fluoxastrobin and ethirimol is molten
Liquid dilutes 1000 times.
Fresh-keeping effects of Fig. 8-HF1 to strawberry biological.Annotation：A, control；B, 0.2% chitosan；C, HF1 and 0.2% first
Shell element.
Growth improvement results of Fig. 9-HF1 to broiler chicken.
The systematic evolution tree of HF1s of the Figure 10-based on 16S rDNA.Annotation：Different plant species 16S rDNA sequences are from NCBI numbers
It is downloaded according to library.B.atrophaeus JCM 9070, NR_024689.1；B.carboniphilus JCM9731, NR_
024690.1；B.flexus IFO15715, NR_024691.1；B.halodurans ATCC 27557, NR_112056.1；
B.lentus NCIMB8773, NR_040792.1；B.marinus, AB021190.1；B.mojavensis IFO15718, NR_
024693.1；B.mycoides 273, NR_036880.1；B.niacini IFO15566, NR_024695.1；
B.psychrosaccharolyticus ATCC 23296, NR_040793.1；B.vallismortis DSM, NR_
024696.1；B.weihenstephanensis DSM, NR_024697.1；B.amyloliquefaciens BG11,
KF699870.Following 16S rDNA sequences come from genome sequence.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.
The relationship of biomass (OD) and inhibition zone size of Figure 11-bacterial strain, in an oscillator, HF1 is in Endo culture mediums
Culture is measured its OD value and while being given birth to fungi using the cylinder plate method measurement of the zymotic fluid after sterilizing in different incubation times
Long inhibiting effect.Fungi is Alternaria alternate opportunistic pathogen (Alternaria alternata).
Embodiment
The separation of 1-HF1 of embodiment and Molecular Identification
Bacterial strain HF1 is separated from cucumber (Cucumis sativus) rhizosphere soil.
According to the genome annotation of HF1, contig 15 (1627bp) is 16S rDNA sequences, and the sequence is as follows：
Based on the comparison of the 16S rDNA sequences carried out with basic Local Alignment Search Tool (BLAST), find HF1's
The 16S rDNA sequence very high homologies of 16S rDNA sequences and bacillus subtilis Pseudomonas, such as bacillus subtilis subspecies
Subtilis str.168 (100%), the bacterial strain are bacillus subtilis reference cultures.Then by using adjacent method
MAGE6.0 rebuilds chadogram (referring to Figure 10), shows that HF1 belongs to bacillus subtilis Pseudomonas.Finally, we order this bacterial strain
Entitled bacillus subtilis HF1, referred to as HF1.
The characteristic of embodiment 2-HF1
HF1 is the bacterium of gram-positive, aerobic formation spore, LB (10g/L peptones, 5g/L yeast powders,
10g/L NaCl) colonial morphology cultivated on agar medium is irregular, has lobate edge (Fig. 1).
HF1 is very sensitive to many antibiotic, such as chloramphenicol, kanamycins, gentamicin, spectinomycin, rifampin, ammonia
Benzyl mycin, tetracycline, streptomysin, penicillin and carbenicillin etc..
At 28 DEG C, under 170rpm, in the 500mL flasks with 200mL LB liquid mediums, the initial OD of HF1600Value
It is 0.267 ± 0.0015.After incubation in 24 hours, the OD of the HF1 of HF1600Reach 5.76 ± 0.03 (Fig. 2).
Preferred culture mediums of the embodiment 3.- for the large-scale production of HF1
It is found that the preferred culture medium of HF1, is named as " Endo ", contain glucose 10-28g/L, KNO3 6g/
L、KH2PO4 1.2g/L、MgSO4·7H2O 1.2g/L、Na3C6H5O7 0.2g/L、CaCl2·2H2O 105mg/L、FeSO4·
7H2O 16mg/L、EDTA2.1mg/L、H3BO3 2.86mg/L、ZnSO4·7H2O 0.222mg/L、MnCl2·4H2O
1.81mg/L、Na2MoO40.021mg/L and CuSO4·5H2O 0.07mg/L.HF1 is containing 10g/L and 28g/L grapes respectively
Growth conditions in the Endo culture mediums of sugar are similar with LB culture mediums, but the yield of HF1 is LB cultures under the same conditions
Twice (Fig. 2) of base.After the Endo medium culture HF1 thalline 24 hours respectively containing 10g/L and 28g/L glucose,
OD600Value may respectively be 11.22 ± 0.04 or 11.46 ± 0.11.
Embodiment 4-HF1 has broad spectrum antibiotic activity, this shows that HF1 is important, is potential, for inhibiting plant disease
The biological control agent of disease
By different fungal plant pathogens be seeded in respectively solid PDA medium (potato dextrose agar,
200g/L potatos, 20g/L glucose, 10g/L agar) on, and cultivated about 4 days at 28 DEG C.With card punch with identical life
The edge of the bacterium colony of long ability punches, and obtains the different agar disks with mycelia of a diameter of 0.5cm, agar disks are inoculated into and are contained
There is the center of the culture dish of PDA cultures.Then in the 1 μ L HF1LB culture fluids of place inoculation apart from agar disks about 2cm, training
The inhibition of HF1 is observed after supporting 5 days.
According to cultivation results, the mycelia that HF1 can inhibit many soil to pass nematophyte disease fungus grows (Fig. 3), such as
Fusarium spp.、Colletotrichum spp.、Rhizoctonia spp.、Phytophthora parasitica、
Alternaria spp.、Gaeumannomyces graminis、Verticillium dahliae、Calonectria
nivale、Ceratobasidium cornigerum、Botryosphaeria berengeriana、Stemphylium
solani、Coniothyrium spp.、Phoma crystallifera、Myrmecridium schulzeri、
Corynespora cassiicola and Magnaporthe grisea.
Under experimental conditions, due to Phytophthora nicotianae (Phytophthora nicotianae) and aspergillus flavus
(Aspergillus flavus) growth is too fast, and does not observe the bacteriostatic activity of zymotic fluid, although inventor thinks in higher
Concentration under HF1 zymotic fluids can show to inhibit the activity of both bacterial strains.
In another independent experiment, under the conditions of used, zymotic fluid does not inhibit the life of Escherichia coli (E.coli)
It is long.
The result shows that HF1 may be used as biological control agent or soil-repairing agent to inhibit soil-borne disease.
Embodiment 5-HF1 zymotic fluids have the activity for inhibiting mycelial growth
In order to assess the depression effect that the HF1 zymotic fluids from 2- days LB liquid medium cultures grow fungi, into
Row Oxford cup is tested.Prepare the experiment of opposite culture according to embodiment 4.
At 170rpm and 28 DEG C, culture HF1 about 2 days.After centrifugation, supernatant is collected, then a part of supernatant exists
It sterilizes 10 minutes at 121 DEG C, remaining part is filtered with biofilter (0.22 μm of diameter).
Oxford cup is placed on culture medium, the 100 above-mentioned zymotic fluids of μ l are added in each Oxford cup.After about 4 days, knot can be recorded
Fruit (Fig. 4).As a result display is inhibiting hypha,hyphae by the LB cultivation and fermentations liquid of high-temperature sterilization or the HF1 of biofiltration degerming
There is remarkable effect in terms of growth.
The zymotic fluid of embodiment 6-HF1 can inhibit fungus spore germination
Fungi Alternaria alternate opportunistic pathogen is cultivated about 5 days on PDA solid mediums.To be added in culture dish (9cm) 5ml without
Bacterium water, scrapes mycelia, washes with water, and then obtains spore by filtering, density is about every milliliter of 1,000 spore.
If embodiment 5 prepares 1ml HF1LB zymotic fluids, it is added into 100ml or 500ml PDA culture mediums.Then, will
HF1 zymotic fluids dilute 100 times and 500 times respectively.About 0.2ml spore suspensions are layered in PDA solid medium tablets.
As a result, it has been found that (Fig. 5), the spore that 100 times of diluted HF1 zymotic fluids can completely inhibit Alternaria alternate opportunistic pathogen is sprouted
Hair.However, high-temperature sterilization, 500 times of diluted HF1 zymotic fluids inhibiting rate (being calculated with following formula 1) be 80.25 ±
3.57%, it is higher than filtered, 500 times of diluted HF1 zymotic fluids inhibiting rate (about 20.45 ± 2.43%).
Formula 1
Annotation：CK, the colony counts in control；T, the colony counts in processing group.
Embodiment 7-HF1 may be used as inhibiting some soil-borne diseases and improving the soil-repairing agent of crop yield
In order to investigate effects of the HF1 as soil-repairing agent, we have selected target medicinal plant radix pseudostellariae.Radix pseudostellariae
Soil-borne disease, such as by Fusarium oxysporum (Fusarium oxysporum) and Rhizoctonia solani Kuhn (Rhizoctonia solani)
Therefore caused root rot and the damping-off caused by Rhizoctonia solani Kuhn (R.solani) can carry out continuous plantation and become in soil
It obtains very serious.
Soil with serious soil-borne disease is used as experimental plot.These experimental plots are divided into 10 trial zones.One by 5
The group of a trial zone composition, with the HF1 fermentation liquor treatments with living cells, other trial zones are as a contrast.Each trial zone is (about
2m long, 1.5m wide) 300 plants of seedling of plantation.The record death rate in harvest (see following formula 2).
Seed impregnates about 2min in October diluted with 100 times HF1 zymotic fluids with living cells, then in air
In dry.Then in the soil by seed kind.Plant emergence after, every plant of seedling about 20ml, 300 times it is diluted, with work
The HF1 zymotic fluids of cell pour.Wait for that next spring comes interim, seedling starts to grow, with about 20ml, 300 times it is diluted, carry
The HF1 zymotic fluids of living cells pour each plant.And it compares and then 20ml water is used to pour each plant.
Formula 2
Annotation：CK, the plant quantity of control group；T, the plant quantity of processing group
In experimental plot, the death rate of processing group and control group is 28.29 ± 6.39% and 63.45 ± 8.56% respectively.
The storage root of processing group is bigger than control group (Fig. 6), and the mean fresh of processing group and control group is per hectare respectively
343.45 ± 88.35kg of 958.05 ± 89.35kg and per hectare.The result shows that HF1 can be a kind of potential inhibition soil biography
Disease and the soil-repairing agent for improving crop yield.
Embodiment 8-HF1LB zymotic fluids can inhibit powdery mildew
HF1 is cultivated in LB liquid medium.Zymotic fluid with living cells is referred to as " HF1LB zymotic fluids ".
HF1LB zymotic fluids with living cells carry out 300 times of dilutions, are applied as foliar spray.
1,000 times of diluted Fluoxastrobins or ethirimol are as positive control.Water is as negative control (CK).
It is primary per 5-7 days foliar sprays by 100 plants of plants for handling, about 3 times altogether.Disease scale is as follows：0 grade, do not have
Spot；1 grade, the ratio of susceptible area and total leaf area is less than 25%；3 grades, the ratio of susceptible area and total leaf area is 25%-
50%；5 grades, the ratio of susceptible area and total leaf area is 50%-75%；7 grades, the ratio of susceptible area and total leaf area is more than
75%.Disease index (%)=∑ (quantity of disease series × respective vanes)/(investigation blade amt × highest disease series)]
× 100%.Control effect (%)=[1- (disease index of disease index/control of processing group)] × 100%.
In the greenhouse, HF1LB zymotic fluids are to powdery mildew of cucumber (Sphaerotheca fuliginea (Schlecht)
Poll control effect) is about 89.46 ± 3.65%, and positive control is about 91.26 ± 4.11%.For pea, band
It is about 81.23 ± 2.31% to the control effect of powdery mildew (Erysiphe pisi DC) to have the HF1LB zymotic fluids of living cells,
Positive control is about 84.98 ± 3.71%.The results show that the HF1LB with living cells can inhibit powdery mildew.
Embodiment 9-HF1LB zymotic fluids may be used as bio-preservative.
In order to investigate the biological preservation effect of HF1, HF1 zymotic fluids are heated into about 10min at 100 DEG C, then 12,
000rpm is centrifuged.Collect supernatant, and 100 times of dilutions.Using perishable strawberry as target fruit.Strawberry is respectively in sterile water
About 20- is impregnated in (negative control), 0.2% chitin or 100 times of diluted HF1LB zymotic fluids containing 0.2% chitin
It 30 seconds, then dries.Processed fruit is mounted in crisper at room temperature.Each processing with 10 strawberries, and each
Processing is repeated 3 times.
Five days after processing, the strawberry of negative control, 0.2% chitin and the HF1LB zymotic fluids containing 0.2% chitin is mouldy
Rotten ratio is respectively 100%, 82.35 ± 4.32% and 13.45 ± 2.45% (Fig. 7).
Therefore, HF1LB zymotic fluids can be used as bio-preservative.
Embodiment 10-HF1 zymotic fluids can improve the growth of target animal as feed addictive.
The antibiotic of low dosage can improve the weight of animals, but it can severely impact animal, the final health for influencing people,
And it also pollutes the environment.
It needs for replacing the safer of antibiotic, the smaller additive of harmfulness in animal feeding.
In China, bacillus subtilis has been authorized the feed addictive as white meat-type chickens by the Ministry of Agriculture.With negative control
It compares, is 3.5% by using the final weight gain that conventional bacillus subtilis strain obtains, raised with antibiotic not apparent
Difference.
According to the experimental result of invention described herein people, low dosage (0.1%-1% is added in drinking water) is found
HF1 can significantly improve the weight gain of white meat-type chickens.
In first experiment, HF1 is cultivated in LB and Endo culture mediums respectively.By zymotic fluid after 10,000rpm centrifugations
It is heated at 100 DEG C about 10 minutes.
LB zymotic fluids are diluted with water to 1%, 0.5% and 0.25%, Endo zymotic fluids and are also diluted with water to 1%.
From the 1st day to the 42nd day, using diluted zymotic fluid as broiler chicken (Gallus gallus domesticus
Baiyu daily drinking water), is used in combination basal feed to raise table hens.Edible basal feed simultaneously drinks the water without any additive
Broiler chicken is as negative control (CK)；The broiler chicken of the basal feed of the edible virginiamycin containing 15mg/kg is as positive control.
Each processing includes 6 groups, every group of 10 chickens.Every 7 days, the weight of chicken is recorded before 8 feedings.
When the off-test at the 42nd day, drink 1%, 0.5%, the chicken of 0.25%LB zymotic fluids and 1%Endo zymotic fluids
Average weight be respectively 2771.90 ± 19.06g, 2698.38 ± 13.92g, 2651.55 ± 6.28g and 2775.23 ±
39.69g。
The average weight of CK and positive control is 2309.90 ± 26.39g and 2331.95 ± 16.60g respectively.
Compared with CK, drink 1%, 0.5%, the body weight growth rate of the chicken of 0.25%LB zymotic fluids and 1%Endo zymotic fluids
It is 20.01 ± 1.85%, 16.82 ± 1.79%, 14.80 ± 1.73%, 20.15 ± 1.80% respectively.
Compared with positive control, drink 1%, 0.5%, the weight of the chicken of 0.25%LB zymotic fluids and 1%Endo zymotic fluids
Growth rate is 13.73 ± 1.33%, 10.71 ± 1.24%, 8.79 ± 1.16% and 13.86 ± 1.26% respectively.
Anatomical results show, with compare and antibiotic treatment compared with, the liver of chicken, heart, spleen, lung, kidney, 12
Do not note abnormalities symptom in duodenum 12 and jejunum.Also without finding any exception in blood biochemical detection.
However, it is elongated to attenuate through the intestinal villus in processing animal duodenum, it can be found that more acini lienalis.It is not intended to
It is bound by theory, these changes may be such that the health of chicken benefits, therefore have weightening effect.
In other experiments, at 42 days, the weight of HF1 processing groups was apparently higher than the body of negative control and positive control
Weight：Compared with negative control, weight gain 28%, with compared with existing commercial bacillus subtilis Probiotic supplement, body
Increase by 16% again, compared to different antibiotic (virginiamycin, Amoxicillin), weight gain 25%.
Furthermore, it was demonstrated that when the solid (starch or rice husk/starch mixture) with the HF1 cultures added with dried forms
When raising chicken, also show beneficial activity (result is not shown).
It to animal is safe that embodiment 11- tests HF1 by acute toxicity test.
In order to study HF1 acute toxicities, select the CD-1 mouse of 20 ± 2g as target animal.Mouse is divided into 5 groups.Often
Organize totally six mouse (3 male mouse and 3 female mices).Mouse stops feeding in experiment the previous day.By a gavage to each group
0.4ml 0.8%NaCl, 10 are applied respectively9cfu/mL、1011cfu/mL、1012It the HF1LB zymotic fluids of cfu/mL and does not dilute
HF1LB zymotic fluids.Then it normally feeds mouse about 15 days, all mouse is killed after 15 days.Observe the clinical manifestation of mouse.
At the end of experiment, all test mouse all live.The clinical manifestation of the mouse of test with compare (0.8%NaCl groups)
Compared to what no difference.Clinical manifestation includes daily feed, drinking-water, hair color, excrement, weight, the death rate and general dissection knot
Fruit.Find there is no visible abnormal structure in all test mices according to anatomical results.Acute toxicity test shows, bud
Spore bacillus strain HF1 is safe for mammal.
It prints off (original document is electrical form)
(table not as and be also not considered the table of international application)
Claims (55)
1. a kind of accession number is bacillus subtilis (Bacillus subtilis) isolated strains HF1 of CGMCC No.11487
Or its mutant strain.
2. the spore of bacillus subtilis isolated strains described in claim 1.
3. the cultivation and fermentation liquid of bacillus subtilis isolated strains described in claim 1.
4. a kind of method of culture bacillus subtilis isolated strains described in claim 1, the method includes making described point
It grows from bacterial strain, is cultivated 1-4 days optionally under 15-37 DEG C of aerobic conditions in suitable culture medium.
5. a kind of method producing bacillus subtilis isolated strains described in claim 1, the method includes implementing right
It is required that the method described in 4, and further comprise recycling bacillus cell from culture medium.
6. the method described in claim 5, wherein passing through one or more carry out institutes in washing, filtering and/or sedimentation
State recycling.
7. method described in claim 5 or 6, wherein by the cell recycled preservation under low temperature or lyophilisation condition.
8. by or can pass through the method described in any one of claim 5-7 obtain B. subtilis cell.
9. the cell extract of the bacillus subtilis isolated strains described in claim 1 or 8, cell suspension, cell homogenates, thin
Cellular lysate liquid or cell mass.
10. a kind of composition, the composition is included in the bacillus subtilis described in claim 1 in suitable carrier, power
Profit requires the spore described in 2, the cultivation and fermentation liquid described in claim 3, cell according to any one of claims 8 or claim 9
The cell extract, cell suspension, cell homogenates, cell pyrolysis liquid or cell mass or the composition include suitable
Carrier, optionally, the composition exist in a dry form.
11. composition according to any one of claims 10, the composition includes bacillus subtilis HF1 intact cells, a concentration of 1 ×
103To 1 × 1014。
12. a kind of method producing bacillus subtilis HF1 supernatant prepared products, the method includes implementing claim 4 institute
The method stated simultaneously further comprises recycling supernatant from culture medium.
13. the method described in right 12, wherein described in one or more progress in washing, filtering and/or sedimentation
Recycling.
14. the method described in claim 13, wherein the supernatant prepared product is recovered by centrifugation.
15. the method described in any one of claim 12-14, wherein supernatant prepared product is heated at least 100 DEG C.
16. the method described in any one of claim 12-15, wherein supernatant prepared product are filtered with biofilter,
The diameter of biofilter is equal to or more than 0.22 μm.
17. the method described in any one of claim 12-16, wherein supernatant prepared product is diluted to 0.1%-5%.
18. a kind of bacillus subtilis HF1 supernatant prepared products, by or can pass through any one of claim 12-17 institutes
The method stated obtains, and the supernatant prepared product is optionally dried forms.
19. a kind of method producing bacillus subtilis HF1 preparations, the method includes from bacillus subtilis HF1 raw material
Middle separating active substances, the raw material are the cells described in supernatant prepared product or claim 9 described in claim 18
Extract, cell suspension, cell homogenates, cell pyrolysis liquid and cell mass,
The method includes using to be selected from selective precipitation, proteolysis, ultrafiltration, ion-exchange chromatography, positive or reverse phase color
One or more methods in spectrometry are classified, and are then measured component and are inhibited the mycelia growth of Soil-born plant pathogenic fungi or change
The growth of kind animal or the ability of health.
20. the bacillus subtilis HF1 preparations selected from following list are used to control the purposes of plant disease：
(i) bacillus subtilis isolated strains described in claim 1；
(ii) spore described in claim 2；
(iii) the cultivation and fermentation liquid described in claim 3；
(iv) cell described in claim 9；
(v) cell extract according to any one of claims 10, cell suspension, cell homogenates, cell pyrolysis liquid or cell mass；
(vi) composition described in claim 10 or 11；
(vii) the bacillus subtilis HF1 supernatant prepared products described in claim 18, selectively, supernatant prepared product quilt
It partly purifies, be classified or concentrate.
21. a kind of method for treating or controlling plant disease, the method includes making described in plant and claim 20
Hay bacillus HF1 preparations contact.
22. the method described in claim 21, wherein the disease is by soil or airborne disease.
23. the method described in claim 21 or 22, wherein applied the preparation by preparation is applied to soil
In plant, the soil species is implanted with plant or i.e. by planting plants.
24. the method described in claim 21 or 22, wherein the preparation is applied to vegetable seeds.
25. the method described in claim 21 or 22, wherein by spraying, dusting, foliage spray, atomization, aerosolization or fumigate
The preparation is applied to plant.
26. the method described in claim 21 or 22 or 25, wherein the preparation is applied to plant seedlings.
27. the method described in claim 21 or 22 or 25, wherein the plant when preparation is applied to harvest or after harvesting, with
Prevent or reduce to the greatest extent the disease after harvest.
28. the method described in any one of claim 21-27, wherein different times of the preparation in plant life cycle
It applies repeatedly.
29. the method described in any one of claim 21-28, wherein the preparation and other diseases controlling agent be administered in combination in
Plant.
30. a kind of method for keeping agricultural plant product fresh-keeping, the method includes making the withered grass bud described in claim 20
Spore bacillus HF1 preparations are contacted with plant product.
31. the method described in any one of claim 21 to 30, wherein the plant is selected from cereal, vegetables and cultivable work
Object, grass, lawn, pasture, fruit tree, ornamental trees and plant, flowering plant, medicinal plant, tobacco.
32. the method described in any one of claim 21 to 31, wherein the disease is fungus-caused by phytopathogen
Disease.
33. a kind of method for controlling plant pathogenic fungi, the method includes to make bacillus subtilis described in claim 20
Bacterium HF1 preparations and Fungal contact.
34. method of claim 33, wherein the preparation is applied to the environment residing for fungi.
35. the method described in any one of claim 32-34, wherein the preparation inhibits the mycelia growth of fungi or spore to sprout
Hair.
36. the method described in any one of claim 32-35, wherein the plant pathogenic fungi is selected from：Fusarium
spp.、Colletotrichum spp.、Rhizoctonia spp.、Phytophthora spp.、Alternaria
spp.Gaeumannomyces graminis、Verticillium dahliae、Calonectria nivale、
Ceratobasidium cornigerum、Botryospuaeria berengeriana、Stemphylium solani、
Coniothyrium spp、Phoma crystallifera、Myrmecridium schulzeri、Corynespora
cassiicola。
37. a kind of method of soil remediation, the method includes so that the bacillus subtilis HF1 preparations described in claim 20 is connect
Touch soil.
38. a kind of composition, bacillus subtilis HF1 preparations described in claim 20 and agriculturally available are included at least
Carrier.
39. the composition described in claim 38, wherein agriculturally available carrier includes one or more moisturizer, sprawls
Agent, stabilizer, bleeding agent, emulsifier, dispersant, surfactant, buffer or adhesive are pasted in agent.
40. the composition described in claim 38 or 39 can be spraying, suspension, concentrate, foam, immersion liquid, slurries, coagulate
The form of glue, dip or paste.
41. the bacillus subtilis HF1 preparations selected from following list are used to improve the purposes of the weight of animals：
(i) bacillus subtilis isolated strains described in claim 1；
(ii) spore described in claim 2；
(iii) the cultivation and fermentation liquid described in claim 3；
(iv) cell described in claim 9；
(v) cell extract according to any one of claims 10, cell suspension, cell homogenates, cell pyrolysis liquid or cell mass；
(vi) composition described in claim 10 or 11；
(vii) bacillus subtilis HF1 supernatant prepared products described in claim 18, selectively, the supernatant prepared product
It is partly purified, is classified or concentrates；
The wherein described animal is optionally people.
42. the bacillus subtilis HF1 preparations selected from following list are being prepared for improving the weight of animals or animal health
Composition aspect in purposes：
(i) bacillus subtilis isolated strains described in claim 1；
(ii) spore described in claim 2；
(iii) the cultivation and fermentation liquid described in claim 3；
(iv) cell described in claim 9；
(v) cell according to any one of claims 10, cell suspension, cell homogenates, cell pyrolysis liquid or cell mass；
(vi) composition described in claim 10 or 11；
(vii) the bacillus subtilis HF1 supernatant prepared products described in claim 18, selectively, the supernatant system
Standby object is partly purified, is classified or concentrates,
The wherein described animal is optionally people.
43. a kind of method improving growth of animal or health, the animal is optionally people, and the method includes by claim
Bacillus subtilis HF1 preparations described in 41 or 42 are applied to animal.
44. the method described in claim 43, the method compared with control-animal for improving the production through handling animal groups
Parameter, the manufacturing parameter is selected from averagely every daily gain, feed conversion rate and through handling the growth consistency in animal groups.
45. the method described in claim 43 or 44, wherein the preparation is by being administered orally to animal, for improving animal
Intestinal health.
46. the method described in any one of claim 43-45, wherein the animal is poultry or cultivation aquatile, preferably
Fish or Crustaceans.
47. the method described in any one of claim 43-46, wherein the animal is poultry, preferably chicken or turkey, the system
Agent can be added in its drinking water or be added in solid-state feed in a dry form.
48. from the bacillus subtilis HF1 preparations selected in following list answering in improving the method for animal health or weight
With：
(i) bacillus subtilis isolated strains described in claim 1；
(ii) spore described in claim 2；
(iii) the cultivation and fermentation liquid described in claim 3；
(iv) cell described in claim 9；
(v) cell extract according to any one of claims 10, cell suspension, cell homogenates, cell pyrolysis liquid or cell mass；
(vi) composition described in claim 10 or 11；
(vii) the bacillus subtilis supernatant prepared product described in claim 18, selectively, supernatant prepared product portion
It is purified, is classified or concentrates with dividing.
49. the application of the preparation described in claim 48, wherein the method are the sides described in any one of claim 43-47
Method.
50. a kind of human food or other animal feeds or replenishers, it includes the preparations described in claim 48, optionally
For the feeding additive aquatic animal or solid food additive for fishery, or the additive for drinking water.
51. the application with the growth medium formed as follows in terms of growth, culture or fermentation Bacillus strain：Glucose
10-28g/L、KNO3 6g/L、KH2PO4 1.2g/L、MgSO4·7H2O1.2g/L、Na3C6H5O7 0.2g/L、CaCl2·2H2O
105mg/L、FeSO4·7H2O 16mg/L、EDTA 2.1mg/L、H3BO3 2.86mg/L、ZnSO4·7H2O 0.222mg/L、
MnCl2·4H2O 1.81mg/L、Na2MoO40.021mg/L and CuSO4·5H2O 0.07mg/L。
52. the application described in claim 51, the growth medium is detached for bacillus subtilis strain described in claim 1
Growth, culture or the fermentation of bacterial strain.
53. a kind of method for the mutant strain preparing the bacillus subtilis HF1 isolated strains that accession number is CGMCC No.11487,
The method includes carrying out luring prominent processing to the bacterial strain.
54. the method described in claim 53, wherein the mutagenic treatment is ultraviolet mutagenesis processing.
55. the method described in claim 53 or 54 is screened wherein after the mutagenic treatment and selects withered grass gemma
The mutant strain of bacillus HF1 bacterial strains, the mutant strain show antiseptic improve or improved and generate or enhance growth of animal.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015094112 | 2015-11-09 | ||
| CNPCT/CN2015/094112 | 2015-11-09 | ||
| PCT/EP2016/077179 WO2017081105A1 (en) | 2015-11-09 | 2016-11-09 | Bacillus strains and agents with beneficial properties |
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| CN108603161B CN108603161B (en) | 2021-11-09 |
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| WO (1) | WO2017081105A1 (en) |
Cited By (3)
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| CN111316998A (en) * | 2020-03-03 | 2020-06-23 | 胡赞民 | Application of bacillus subtilis HF1 in planting rosaceous plants |
| CN111801020A (en) * | 2018-02-02 | 2020-10-20 | 诺维信公司 | Management of pathogenic Lawsonia |
| CN112438171A (en) * | 2019-09-02 | 2021-03-05 | 北京中科金株生物科技有限公司 | Application of bacillus subtilis HF1 in astragalus planting |
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| BR112018067860B1 (en) | 2016-02-29 | 2023-04-18 | United Animal Health, Inc. | DIRECTLY FED MICROBES |
| WO2019040266A1 (en) * | 2017-08-23 | 2019-02-28 | Novozymes A/S | Direct fed microbials for improving the general condition and health of fish |
| TW201915165A (en) * | 2017-09-20 | 2019-04-16 | 行政院農業委員會農業藥物毒物試驗所 | Bacillus subtilis strain for preparing fermented feather meal and use thereof for producing feather powder and proteolytic enzyme and keratinolytic enzyme strains |
| CN107881128B (en) * | 2017-10-27 | 2020-08-25 | 湖北省农业科学院中药材研究所 | Strain BZJN1 for preventing and treating root rot of bighead atractylodes rhizome and preparation method and application thereof |
| US11627741B2 (en) | 2018-09-28 | 2023-04-18 | Microbial Discovery Group, Llc | Microorganisms for plant pathogen inhibition |
| WO2020072578A1 (en) | 2018-10-02 | 2020-04-09 | Microbial Discovery Group, Llc | Microbials for animals |
| CN110564655A (en) * | 2019-10-17 | 2019-12-13 | 中国烟草总公司陕西省公司 | Preparation and application of bacillus subtilis SL-19 microbial inoculum for biological control of alternaria alternate |
| CN114836333A (en) * | 2021-02-01 | 2022-08-02 | 武汉渔庆家电子商务有限公司 | Blue algae purifying agent and preparation and application thereof |
| CN113174352B (en) * | 2021-04-09 | 2022-04-29 | 中国科学院遗传与发育生物学研究所 | Bacillus subtilis HF1 mutant and construction method and application thereof |
| CN114058555B (en) * | 2021-12-13 | 2023-09-05 | 山西农业大学 | Preparation and application methods of biological seed coating agent for preventing and treating corn stem rot |
| CN114480204B (en) * | 2022-02-18 | 2023-04-25 | 安徽科技学院 | Bacillus nicotinate FY2 and application thereof |
| CN114806966B (en) * | 2022-05-20 | 2023-02-28 | 河北农业大学 | Pesticide-resistant Kulas-resistant wheat holoetch disease biological control bacillus subtilis and application thereof |
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| WO2017081105A1 (en) | 2017-05-18 |
| CN108603161B (en) | 2021-11-09 |
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