CN109321500A - One bacillus amyloliquefaciens bacterial strain and its application in prevention and treatment Oil Tea Anthracnose evil - Google Patents
One bacillus amyloliquefaciens bacterial strain and its application in prevention and treatment Oil Tea Anthracnose evil Download PDFInfo
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- CN109321500A CN109321500A CN201811210897.5A CN201811210897A CN109321500A CN 109321500 A CN109321500 A CN 109321500A CN 201811210897 A CN201811210897 A CN 201811210897A CN 109321500 A CN109321500 A CN 109321500A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention discloses a bacillus amyloliquefaciens bacterial strains, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain is preserved in Guangdong Province's Culture Collection, it is named as bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10), the deposit number of Culture Collection is GDMCC No:60394 in Guangdong Province.The bacterial strain significantly inhibits Oil Tea Anthracnose opportunistic pathogen bacterium, can be good at being applied to prevention and treatment Oil Tea Anthracnose evil, has the potentiality for promoting plant growth, be a kind of very strong microbial strains of adaptability.The invention also discloses application of the bacterial strain in prevention and treatment Oil Tea Anthracnose evil, the effective solution substitution of chemical agent reduces pesticide residue, ensure that the quality safety of agricultural product, has certain practical value and application value.
Description
Technical field
The invention belongs to microbial engineering field more particularly to a bacillus amyloliquefaciens bacterial strain and its preventing and treating
Application in Oil Tea Anthracnose evil.
Background technique
Oil tea (Camellia Oleifera Aabel) is Theaceae Camellia Plants, originating from China, is had long
Cultivation history is the world four big woody edible oil source tree species one of eponymous with oil palm, olive and coconut.The oil tea resource in China
It is abundant, it is mainly distributed on the provinces and regions such as southern Hunan, Jiangxi, Guangxi, Zhejiang, Fujian, Anhui, Guizhou.According to statistics, national at present
Having camellia oleifera lam is about 4,000,000 hm2To produce 600000 tons of tea seed per year.Influence of the oil tea disease to its yield also becomes increasingly conspicuous, wherein
Oil Tea Anthracnose is one of most important disease, often results in that oil tea blade is withered, fruit is caducous, fruit drop rate is generally 20%~
40%, up to 60% or more when serious, so as to cause the oil tea underproduction 10%~30%, even up to 40%~50%, cause huge
Huge economic loss, Oil Tea Anthracnose have become the important potential threat of camellia oleiferaindustry.Therefore, it is necessary to reinforce to Oil Tea Anthracnose
Research dynamics, improve to the Prevention Technique of Oil Tea Anthracnose.
It is directed to Oil Tea Anthracnose, common control measure has chemical pesticide sprinkling, but chemical method pollution is larger.It needs
The method for seeking more environmentally friendly microbial control seems increasingly important, and biological control is not easy to keep pathogen generation anti-due to it
The low hot spot for being increasingly becoming everybody and paying close attention to and studying of pharmacological property, environmentally safe, active high and production cost.Such as: Meng Qingmin is ground
Studying carefully discovery oil tea Endophytic antagonistic bacteria Y13 is one plant of Bacillus subtillis (oil having to Oil Tea Anthracnose bacterium compared with high inhibition effect
The main antibacterial substance of tea anthracnose antagonistic bacterium Y13 isolates and purifies and the mode of action, plant protection, 02 phase in 2014), believe coral coral
Deng being lipopeptid with the antibacterial substance in bacillus amyloliquefaciens WH1 fermentation liquid, which can make Oil Tea Anthracnose mycelia
The fermentation liquid of deformity, highly diluted multiple still has significant preventive effect (1 plant of solution starch gemma to the anthracnose of in vitro Camellia Leaves on piece
The optimization of bacillus fermentation condition and its to the preventive effect Hua Zhong Agriculture University journal of Oil Tea Anthracnose, 04 phase in 2011).But it is above-mentioned
The tolerance of kind is poor, and adaptability is poor.Therefore, find that a kind of tolerance is more preferable, more adaptable can be used for preventing and treating oil tea
The strain of anthracnose has a very important significance this field.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the shortcomings of to mention in background above technology and defect, provide one
Bacillus amyloliquefaciens bacterial strain and its application in prevention and treatment Oil Tea Anthracnose evil.
In order to solve the above technical problems, technical solution proposed by the present invention is to provide a bacillus amyloliquefaciens bacterial strain,
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain is preserved in Guangdong Province's Microbiological Culture Collection
The heart is named as bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10), in Guangdong Province
The deposit number of Culture Collection is GDMCC No:60394, and the preservation time is on June 25th, 2018, depositary institution
Address be located at 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Microbes Inst.
Bacillus amyloliquefaciens can produce protease and cellulase during growth metabolism, moreover it is possible to generate to oil tea anthrax
Sick and other crop diseases have the volatile compound, crude protein and thick lipopeptid for the effect that significantly inhibits, and are with good biological
The microorganism fungus kind of prevention and treatment prospect.
Above-mentioned bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10) is from oil tea
Acquisition is isolated and purified in anthrax region of disease healthy plant rhizosphere soil sample, on NA plate, is inverted 30 DEG C of culture a period of times
Afterwards, colonial morphology is creamy white, translucent, and round, edge is irregular, and surface wettability is smooth, and micro- sem observation cell is in the shape of a rod,
Gram's staining is bluish violet, positive.
Above-mentioned bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10) can produce
Antibacterial substance, including antibacterial albuminoid, lipopeptid class antibiont, oligopeptidase, polyketide etc., these substances all have very
Strong biocidal property.The bacterial strain, which has, simultaneously produces heteroauxin, phosphorus decomposing and produces ammonia ability, has the potentiality for promoting plant growth.
It is grown under conditions of CFC-10 bacterial strain can be 4.0-8.5 in pH, temperature range is 20 DEG C -55 DEG C, can be resistant to 90 DEG C of temperature, say
Bright is that a kind of tolerance is fine, the very strong microbial strains of adaptability.
The DNA base of above-mentioned bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10)
Because of sequence, as shown in SEQ ID NO.1.
Above-mentioned bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10),
16SrDNA sequence carries out homology analysis with BLAST in ncbi database, obtains the higher sequence of similitude, uses
MEGA6.06 software building phylogenetic tree；Bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens
CFC-10 16SrDNA sequence) and the homology of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) reach
99%, therefore it is accredited as bacillus amyloliquefaciens.
Comprehensive morphological observation, Physiology and biochemistry identification and 16SrDNA the sequencing results, can determine the solution starch gemma
Bacillus CFC-10 is bacillus amyloliquefaciens kind (Bacillus amyloliquefaciens), is named as solving starch gemma
Bacillus CFC-10.
Above-mentioned bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10) fermentation liquid pair
The inhibition zone of antagonism Oil Tea Anthracnose opportunistic pathogen has reached 28.97mm, illustrates bacillus amyloliquefaciens CFC-10 provided by the invention
(Bacillus amyloliquefaciens CFC-10) significantly inhibits Oil Tea Anthracnose opportunistic pathogen bacterium, thus
It can be good at being applied to prevention and treatment Oil Tea Anthracnose evil.
Based on a total technical concept, the present invention correspondingly provides the above-mentioned Bacillus amyloliquefaciens strain of one kind anti-
Control the application in Oil Tea Anthracnose evil.
Above-mentioned application, it is preferred that the method for the application specifically comprises the following steps:
(1) the bacillus amyloliquefaciens CFC-10 is seeded in seed culture medium the seed cultivated, obtained
Liquid, which is seeded in fermentation medium, to ferment, and obtains bacillus amyloliquefaciens CFC-10 zymocyte liquid；
(2) Oil Tea Anthracnose early stage and before, using the bacillus amyloliquefaciens obtained after the step (1)
Oil tea trunk is sprayed on after the dilution of CFC-10 zymocyte liquid.
Preferably, in the step (1), the component of seed culture medium includes beef extract, peptone and sodium chloride, the ox
The mass ratio of meat extract, peptone and sodium chloride is (2.3-3.1): (8.5-10): (3.8-5.1).
Preferably, in the step (1), the component of fermentation medium includes corn flour, soybean powder and CaCl2, the jade
Rice flour, soybean powder and CaCl2Mass ratio be (10-20): (6-15): (1-3).
Preferably, in the step (1), the temperature of culture is 28-32 DEG C, incubation time 18-24h, seed culture medium
PH is in 7.0-7.2.
Preferably, in the step (1), the inoculum concentration of fermentation is 0.1%-5%, 25-45 DEG C of fermentation temperature, fermentation time
In 7.0-8.0, the revolving speed of shaking table concussion is 150-250rpm by 32-48h, the pH of fermentation medium.
Preferably, in the step (2), bacillus amyloliquefaciens CFC-10 zymocyte liquid is diluted with water, strain after dilution
Concentration is 108Cfu/mL or more.
Preferably, in the step (2), spraying method is at least to spray 2 times, between spraying every time to squirt trunk as degree
Every 8-10d.
Compared with prior art, the invention has the benefit that
1, Bacillus amyloliquefaciens strain provided by the invention is bacillus amyloliquefaciens CFC-10, and the bacterial strain is to oil tea charcoal
Subcutaneous ulcer pathogen bacterium significantly inhibits, and can be good at being applied to prevention and treatment Oil Tea Anthracnose evil, which can also produce
Raw heteroauxin has dissolving P capacity, produces ammonia ability, has the potentiality for promoting plant growth；The bacterial strain can be 4.0- in pH
8.5, temperature range is grown under conditions of being 20 DEG C -55 DEG C, be can be resistant to 90 DEG C of temperature, is a kind of very strong microbial bacteria of adaptability
Strain.
2, application of the invention, Oil Tea Anthracnose early stage and before, using bacillus amyloliquefaciens CFC-10 send out
Oil tea trunk is sprayed on after the dilution of yeast-like fungi liquid, can be good at preventing and treating Oil Tea Anthracnose evil, effective solution chemical agent
Substitution, reduces pesticide residue, ensure that the quality safety of agricultural product, has certain practical value and application value.
One bacillus amyloliquefaciens bacterial strain is named as bacillus amyloliquefaciens CFC-10 (Bacillus
Amyloliquefaciens CFC-10), it is preserved in Guangdong Province's Culture Collection (abbreviation GDMCC), in Guangdong
The deposit number for saving Culture Collection is GDMCC No:60394, and the preservation time is on June 25th, 2018, preservation list
The address of position is located at 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Microbes Inst.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the colonial morphology figure of bacillus amyloliquefaciens CFC-10 in embodiment.
Fig. 2 is the Gram's staining figure of bacillus amyloliquefaciens CFC-10 in embodiment.
Fig. 3 is the systematic growth tree graph of the 16S rDNA sequence construct of bacillus amyloliquefaciens CFC-10 in embodiment.
Fig. 4 be in embodiment bacillus amyloliquefaciens CFC-10 to the antibacterial figure of Oil Tea Anthracnose opportunistic pathogen.
Specific embodiment
To facilitate the understanding of the present invention, the present invention is done below in conjunction with Figure of description and preferred embodiment more complete
Face meticulously describes, but protection scope of the present invention is not limited to following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter are generally understood meaning phase with those skilled in the art
Together.Technical term used herein is intended merely to the purpose of description specific embodiment, and it is of the invention to be not intended to limitation
Protection scope.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment:
Bacillus amyloliquefaciens bacterial strain provided by the invention, is named as bacillus amyloliquefaciens CFC-10
(Bacillus amyloliquefaciens CFC-10) is preserved in Guangdong Province's Culture Collection (referred to as
It GDMCC), is GDMCC No:60394 in the deposit number of Guangdong Province's Culture Collection, the preservation time is 2018
In on June 25, in, the address of depositary institution is located at 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Province's microbe research
Institute.
1, the screening of bacterial strain
S01: oil tea anthrax region of disease healthy plant rhizosphere soil sample is acquired by multi-point sampling method, and will be collected
Soil sample is finely ground；
S02: finely ground soil sample is put into the centrifuge tube equipped with sterile water and is fullyd shake, Soil Slurry is obtained；
S03: by the Soil Slurry gradient dilution, the Soil Slurry of various concentration is obtained；
S04: choosing concentration is 10-5、10-6With 10-7Soil Slurry, and be added on NA culture medium flat plate and applied
Cloth processing, is put into 30 DEG C of incubator and cultivates, obtain bacterium colony；
S05: selecting the different single colonie of form and carry out scribing line conservation, cultivate 24 hours, separation bacterium is obtained, by the separation
Bacterium is placed in 4 DEG C of refrigerator and saves backup；
S06: being inoculated in PDA culture medium plate center for Oil Tea Anthracnose opportunistic pathogen fungus block with the punch of diameter 0.5cm,
The different bacillus of equidistant inoculation at the 2.5cm right-angled intersection of anomaly plate center is control not to be inoculated with oil tea anthrax strain,
3 repetitions of every processing；25 DEG C of cultures, when full ware is covered in control, record has the bacterium of bacteriostasis, and it is maximum to obtain inhibition zone
Strain is bacillus amyloliquefaciens CFC-10.
2, the identification of bacterial strain
The bacterial strain that the present invention provides embodiment is identified that the identification includes the following contents:
1) Morphological Identification
Bacterial strain provided in an embodiment of the present invention is crossed on NA culture medium flat plate, is then reversed plate, is in temperature
The growing state for observing and recording bacterium colony on plate for 24 hours is cultivated under conditions of 30 DEG C.Colonial morphology figure is as shown in Figure 1, bacterium colony is in
Irregular subcircular, edge are irregular, milky, translucent, surface is smooth wet.
Further, with kit Gram's staining is carried out to bacterial strain provided in an embodiment of the present invention and in oily microscopic observation bacterium
Strain and take pictures to bacterial strain, the Gram's staining of the bacterial strain is as shown in Figure 2.After Gram's staining, the embodiment of the present invention is mentioned
The bacterial strain of confession is rod-shaped and purple, is gram-positive bacteria, and spore staining has gemma generation.
2) Physiology and biochemistry is identified
Catalase experiment: directly the hydrogen peroxide of dropwise addition 3% is observed immediately, is had in the liquid culture (for 24 hours) of bacterial strain
A large amount of bubble producers are the positive, and not generating bubble, person is feminine gender.Bacterial strain generates a large amount of bubbles immediately, and experimental result is the positive.
Oxidizing ferment experiment: for one jiao of extracting waste cleaning filter paper in clean culture dish, it is a little to dip test organisms bacterium colony, and examination is added dropwise
Agent (1% hydrochloride base p-phenylenediamine aqueous solution) one is dripped, and positive immediately gradually deepen by pinkiness, later color.Bacterial strain
Discoloration, for the positive.
Starch Hydrolysis experiment: strain point is connected on starch culture-medium, and a small amount of iodine solution is added dropwise in plate, gently in 30 DEG C of culture 48h
Light rotation, makes iodine solution be uniformly distributed in plate, observes.There is colorless and transparent circle and shows the ability for having hydrolysis starch in periphery of bacterial colonies,
Conversely, not having.Bacterial strain periphery of bacterial colonies has transparent circle generation, can hydrolyze starch, for the positive.
Methyl red MR experiment: the new pure culture to be tried of picking is a little, is inoculated in collective media, is incubated at 30 DEG C, 3~
5 days, culture solution 1ml is taken, the red indicator 1~2 of methylate drips, and the positive takes on a red color, and weakly positive is in pale red, and feminine gender is yellow.Bacterium
Liquid flavescence color, for feminine gender.
VP experiment: the new pure culture of picking is a little, is inoculated in collective media, first in 30 DEG C of culture 2d, culture solution 2.5ml
A- naphthols absolute alcohol solution 0.6ml, then plus 40% potassium hydroxide aqueous solution 0.2ml, shake 2~5min, positive bacteria Chang Li is added
Red is presented, if redfree occurs, being statically placed in room temperature or 30 DEG C of insulating boxs be can determine that if do not shown red still in 2h as yin
Property.Bacterium solution reddens immediately, for the positive.
Gelatin liquefaction experiment: new pure culture is taken, percutaneous puncture-inoculation separately there are two not to be inoculated in about 2/3 depth of gelatin high level
Blank control.3~5d is cultivated at 20 DEG C.Observation daily is as a result, if bacterial growth and gelatin partly or entirely becomes flowable
Liquid, then be that test is positive.It otherwise is feminine gender.Gelatin is liquefied, for the positive.
Nitrate reduction experiment: culture is inoculated in nitrate broth culture medium, 28 DEG C of shaking table culture 3d take 5mL to train
Nutrient solution illustrates that color developing agent is added by kit (Hai Bo Bioisystech Co., Ltd nitrate reduction kit), turned yellow as the positive,
Feminine gender is not changed color as.Bacterium solution turns yellow, for the positive.
Produce hydrogen sulfide experiment: by bacterium percutaneous puncture-inoculation to be checked in lead acetate medium, in 35 DEG C of 24~48h of culture observation knots
Fruit.Culture medium blackening is the positive, does not become negative.Culture medium does not change colour, for feminine gender.
Citrate, which utilizes, tests: the streak inoculation on simon Si Shi citrate medium inclined-plane of picking lawn, and 35 DEG C
Culture 3-7 days.Culture medium is that alkalinity (indicator blue or pink) person is positive, is otherwise feminine gender.Culture medium becomes blue, is
It is positive.
Lecithin activity experiment: with 75% ethyl alcohol by Fresh Egg surface sterilization, and egg is beaten with the tweezers of sterilizing
A hole, incline egg white, and yolk then is sucked out with aseptic straw, is added in the culture medium for being cooled to 50 DEG C or so after melting, mixes
Inverted plate after even, point connect strain, and 30 DEG C of culture 24-48h observations, it is enzyme positive that muddy opaque region person, which occurs, in colony edge.Bacterium
It falls around and apparent muddy opaque region occurs, for the positive.
Malonate utilizes experiment: picking culture 12h lawn is inoculated in malonate culture medium, and 24- is cultivated in 35 DEG C of cultures
48h, for culture medium by green change indigo plant person into the positive, on the contrary is negative.Culture medium becomes blue, for the positive.
Glucose fermentation experiment: this bacterial strain percutaneous puncture-inoculation is picking in temperature in glucose oxidation-fermentation medium on a small quantity
3d is cultivated under conditions of 30 DEG C, observes the variation of culture medium color.If needing to continue to observe 7d, culture medium without color change
Flavescence person is fermented type.In this experiment, culture medium turns yellow, and illustrates this bacterial strain for the positive.
Cellulose decomposition experiment: by strain to be tested streak inoculation in the agar based media for adding 0.8% cellulose powder
On, using the culture medium that is not inoculated with as blank control, 30 DEG C are cultivated 1~2 week, and bacterial strain can be grown on culture medium, experimental result
For the positive；It otherwise is feminine gender.Bacterial strain can slowly be grown, for the positive.
Galactose utilization experiment: it by this strain inoculated in gala sugar culture-medium, is cultivated under conditions of temperature is 30 DEG C
2d, observing bacterium colony growing state if bacterium colony is formed can use galactolipin, conversely, not all right.In this experiment, thallus is raw
It is long, for the positive, illustrate that this bacterial strain can use galactolipin.
Arabinose utilizes experiment: by this strain inoculated in arabinose culture medium, under conditions of temperature is 30 DEG C
2d is cultivated, observing bacterium colony growing state if bacterium colony is formed can use arabinose, conversely, not all right.In this experiment,
Thalli growth illustrates that this bacterial strain can use arabinose for the positive.
Mannose utilizes experiment: by this strain inoculated in sweet dew sugar culture-medium, cultivating under conditions of temperature is 30 DEG C
2d, observing bacterium colony growing state if bacterium colony is formed can use mannose, conversely, not all right.In this experiment, thallus is raw
Long, for the positive, this bacterial strain can use mannose.
D-Fructose utilizes experiment: by this strain inoculated in D-Fructose culture medium, cultivating under conditions of temperature is 30 DEG C
2d, observing bacterium colony growing state if bacterium colony is formed can use D-Fructose, conversely, not all right.In this experiment, thallus is raw
Long, for the positive, this bacterial strain can use D-Fructose.
The experiment of D- xylose utilization: it by this strain inoculated in D- xylose media, is cultivated under conditions of temperature is 30 DEG C
2d, observing bacterium colony growing state if bacterium colony is formed can use D- xylose, conversely, not all right.In this experiment, thallus is not
Growth, for feminine gender, this bacterial strain cannot utilize D- xylose.
14%NaCl growth experiment: by this strain inoculated in the NA fluid nutrient medium of the NaCl containing 14%, in temperature
2d is cultivated under conditions of being 30 DEG C, if fermentation liquid becomes cloudy, can be survived in 14%NaCl solution, if not becoming cloudy,
It cannot.In an experiment, fermentation liquid becomes cloudy, and for the positive, can survive in 14%NaCl solution.
It produces heteroauxin (IAA) test: strain to be tested is inoculated into the TSB culture medium of addition 0.5mmol/L L-Trp
In 30 DEG C of shaking table culture 48h, take culture solution 2mL, 10000r/min is centrifuged 10min, takes supernatant, and every 1mL supernatant adds 2mL
Salkowski reagent, room temperature dark place stand 30min, and using blank cultures as control, generating then explanation if any pink colour has IAA
It generates.There is pink colour generation, produce IAA, for the positive.
Produce ammonia test: by strain inoculated into peptone culture medium, 30 DEG C of shaking table culture 3d take culture solution a little in test tube
In, 5 drop nessler reagents are added dropwise, using blank cultures as control, tan precipitate occur for the positive, it was demonstrated that bacterial strain, which has, produces ammonia
Ability.There is tan precipitate generation, have and produce ammonia ability, for the positive.
Phosphorus decomposing test: by the NBRIP culture medium of strain inoculated to 2% agar of addition, 30 DEG C of culture 3d, periphery of bacterial colonies has
It is colorless and transparent to iris out existing person as the positive, it was demonstrated that the bacterial strain has dissolving P capacity.Bacterial strain periphery of bacterial colonies has colourless transparent circle to occur, and is
It is positive.
To sum up, Physiology and biochemistry qualification result such as table 1.
The Physiology and biochemistry qualification result of 1 bacterial strain of table
| Table feature | Response feature | Table feature | Response feature |
| Contact enzymatic determination | + | Citrate utilizes | + |
| Oxidase assay | + | Lecithin activity measurement | + |
| Starch Hydrolysis measurement | + | Malonate utilizes | + |
| Methyl red MR measurement | - | Cellulose decomposition | + |
| VP experiment | + | Galactose utilization | + |
| Gelatin liquefaction measurement | + | Arabinose utilizes | + |
| Nitrate reduction measurement | + | Mannose utilizes | + |
| Glucose fermentation | + | D-Fructose utilizes | + |
| Produce hydrogen sulfide measurement | - | D- xylose utilization | - |
| 14%NaCl growth | + | 20 DEG C of growths | + |
| PH4 growth | + | 55 DEG C of growths | + |
| PH8.5 growth | + | 90 DEG C of heatproofs | + |
| Produce heteroauxin | + | Phosphorus decomposing | + |
| Produce ammonia | + |
Wherein, "+" be expressed as this bacterial strain have reaction or can use, "-" be shown as this bacterial strain do not react or cannot benefit
With.
1 test result of table illustrates that the bacterial strain can produce heteroauxin, has dissolving P capacity, produces ammonia ability, these growth-promotings
Attribute testing result illustrates that bacterial strain has the potentiality for promoting plant growth.CFC-10 bacterial strain can be 4.0-8.5, temperature model in pH
Enclose be 20 DEG C -55 DEG C under conditions of grow, can be resistant to 90 DEG C of temperature, explanation is that a kind of tolerance is fine, adaptability is very strong micro-
Biological bacterial strain.In addition, bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10) can produce
Antibacterial substance, including antibacterial albuminoid, lipopeptid class antibiont, oligopeptidase, polyketide etc., these substances all have very
Strong biocidal property.
3) the 16SrDNA sequencing of bacterial strain:
The embodiment of the present invention uses health to extract in this bacterial strain for century Biotechnology Co., Ltd's genome extraction kit
DNA.Wherein, said extracted kit includes the PCR reaction system of 50 μ L, and PCR reaction system include 25 μ L 2 ×
Master Mix；The upstream primer of 2.5 μ L；The downstream primer of 2.5 μ L；The dd H2O of 18 μ L；The template DNA of 2 μ L.PCR reaction
Condition are as follows: temperature be 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 0.5min；53 DEG C of annealing 0.5min；72 DEG C of extension 1min；30
Circulation, 72 DEG C of extension 5min, 4 DEG C of preservations.PCR product result is sequenced by Shanghai Sheng Gong bioengineering Co., Ltd, sequencing
As a result as shown in sequence table SEQ ID NO.1.Gained sequence carries out sequence alignment analysis by NCBI-BLAST, uses
MEGA6.06 software building phylogenetic tree (as shown in Figure 3), bacillus amyloliquefaciens CFC-10 (Bacillus
Amyloliquefaciens CFC-10) 16S rDNA sequence and bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens homology) reaches 99%.
It is comprehensive to state morphological observation, Physiology and biochemistry identification and 16S rDNA the sequencing results, it can determine that the bacterial strain is
Bacillus amyloliquefaciens are named as bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-
10)。
3. the effect of bacillus amyloliquefaciens CFC-10
Bacillus amyloliquefaciens CFC-10 obtained above (Bacillus amyloliquefaciens CFC-10) is existed
It ferments under the following conditions: corn flour 10-20g/L, soybean powder 6-15g/L, CaCl21-3g/L, 25-45 DEG C, 150-
250rpm, pH7.0-8.0, inoculum concentration 1%-5%, 5L fermenting pot 32-48h obtain zymocyte liquid, and actual measurement spore concentration is
(4.2±0.61)×1010cfu/mL。
Above-mentioned bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens is detected with face-off flat band method
CFC-10) the bacteriostatic activity of zymocyte liquid, as shown in figure 4, reaching to the inhibition zone of antagonism Oil Tea Anthracnose opportunistic pathogen
28.27mm illustrates bacillus amyloliquefaciens CFC-10 provided by the invention (Bacillus amyloliquefaciens CFC-
10) Oil Tea Anthracnose opportunistic pathogen bacterium is significantly inhibited, it is thus possible to be applied to prevention and treatment Oil Tea Anthracnose evil well.
4. application of the bacillus amyloliquefaciens CFC-10 in prevention and treatment Oil Tea Anthracnose evil
1) culture medium
Seed culture medium: beef extract 0.3g/L, peptone 1g/L, sodium chloride 0.5g/L；
Fermentation medium: corn flour 10-20g/L, soybean powder 6-15g/L, CaCl21-3g/L。
2) preparation of bacillus amyloliquefaciens CFC-10 zymocyte liquid
One ring bacillus amyloliquefaciens CFC-10 of picking, is seeded in the 500mL conical flask containing seed culture medium, pH exists
7.0-7.2, cultivation temperature are 30 DEG C, and incubation time is for 24 hours；Then obtained seed liquor is all seeded to containing fermentation medium
5L fermentor in, inoculum concentration 0.1%-5%, pH are in 7.0-8.0, and 25-45 DEG C of fermentation temperature, the revolving speed of shaking table concussion is 150-
250rpm obtains bacillus amyloliquefaciens CFC-10 zymocyte liquid after the 32-48h that ferments.
3) woodland field experiment effect detection
It is carried out in Oil Tea Anthracnose early stage, biological and ecological methods to prevent plant disease, pests, and erosion area, chemical prevention check plot and sky is respectively set 3 testing sites
The processing of white check plot 3,3 plants of each cell, minizone setting protection row.Biological and ecological methods to prevent plant disease, pests, and erosion area uses bacillus amyloliquefaciens CFC-10 bacterium
Suspension is watered dilution by spraying, and chemical prevention check plot is watered dilution by spraying by 500 times using 75% Bravo.It is to squirt trunk
Degree is administered 2 midfeather 10d altogether.
4) implementation result method is investigated
First time investigation is carried out before first time sprays by the professional standard of oil tea leaf portion disease survey, after second sprays
It carries out within ten days investigating for second.Disease index grade scale is as shown in table 2.When investigation, investigation statistics are carried out with disease incidence, according to
Following formula calculates disease index and control efficiency, as a result such as the following table 3.
2 disease index grade scale of table
| Sick grade | Occurring degree | Represent numerical value |
| 0 grade | Whole strain oil tea young leaves on piece does not have any scab to occur | 0 |
| 1 grade | Young leaves on piece has scab appearance, and incidence of leaf number is less than the 1/4 of the new blade of whole strain | 1.0 |
| 2 grades | Young leaves on piece has scab appearance, and incidence of leaf number is less than the 1/4~1/2 of the new blade of whole strain | 2.0 |
| 3 grades | Young leaves on piece has scab appearance, and incidence of leaf number is less than the 1/2~3/4 of the new blade of whole strain | 3.0 |
| 4 grades | Young leaves on piece has scab appearance, and incidence of leaf number is less than the 3/4 of the new blade of whole strain | 4.0 |
The comparison of 3 control efficiency of table
It can be concluded that, contain 10 from 3 data of table8The prevention and treatment of the zymocyte liquid and comparison medicament Bravo of cfu/mL cell number
Effect, which has reached identical, prevents standard, it was demonstrated that content is 108The strain concentration of cfu/mL or more can reach identical effect
Fruit.Zymocyte liquid can be effectively solved the substitution of chemical agent, reduce pesticide residue, ensure that the quality peace of agricultural product
Entirely, there is certain practical value and application value.
Embodiment described above is merely a preferred embodiment of the present invention, and the simultaneously exhaustion of the feasible implementation of non-present invention.It is right
For persons skilled in the art, any aobvious to made by it under the premise of without departing substantially from the principle of the invention and spirit and
The change being clear to should be all contemplated as falling within claims of the invention.
Sequence table
<110>Sino-South African Forestry University of Science and Technology, Hu'nan Prov. Academy of Forest-Sciences
<120>one bacillus amyloliquefaciens bacterial strains and its application in prevention and treatment Oil Tea Anthracnose evil
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213>bacillus amyloliquefaciens CFC-10 (Bacillus amyloliquefaciens CFC-10)
<400> 1
cgcagtgcgg gtgctataca tgcagtcgag cggacagatg ggagcttgct ccctgatgtt 60
agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120
aaaccggggc taataccgga tggttgtctg aaccgcatgg ttcagacata aaaggtggct 180
tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360
ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420
aacaagtgcc gttcaaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540
gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600
gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720
ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1020
tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080
caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140
aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200
cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260
tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320
atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380
accacgagag tttgtaacac ccgaagtcgg tgaggtaacc tttatggagc cagccgccga 1440
agtgacaagt g 1451
Claims (9)
1. a bacillus amyloliquefaciens bacterial strain, which is characterized in that the bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) bacterial strain is preserved in Guangdong Province's Culture Collection, is named as bacillus amyloliquefaciens
CFC-10 (Bacillus amyloliquefaciens CFC-10), the preservation of Culture Collection in Guangdong Province
Number is GDMCC No:60394, and the preservation time is on June 25th, 2018.
2. a kind of application of Bacillus amyloliquefaciens strain according to claim 1 in prevention and treatment Oil Tea Anthracnose evil.
3. application according to claim 2, which is characterized in that the method for the application specifically comprises the following steps:
(1) the bacillus amyloliquefaciens CFC-10 is seeded in seed culture medium and is cultivated, obtained seed liquor connects
Kind is fermented into fermentation medium, obtains bacillus amyloliquefaciens CFC-10 zymocyte liquid；
(2) Oil Tea Anthracnose early stage and before, using the bacillus amyloliquefaciens CFC-10 obtained after the step (1)
Oil tea trunk is sprayed on after zymocyte liquid dilution.
4. application according to claim 3, which is characterized in that in the step (1), the component of seed culture medium includes ox
The mass ratio of meat extract, peptone and sodium chloride, the beef extract, peptone and sodium chloride is (2.3-3.1): (8.5-10):
(3.8-5.1)。
5. application according to claim 3, which is characterized in that in the step (1), the component of fermentation medium includes jade
Rice flour, soybean powder and CaCl2, the corn flour, soybean powder and CaCl2Mass ratio be (10-20): (6-15): (1-3).
6. application according to claim 3, which is characterized in that in the step (1), the temperature of culture is 28-32 DEG C, training
Time 18-24h is supported, the pH of seed culture medium is in 7.0-7.2.
7. application according to claim 3, which is characterized in that in the step (1), the inoculum concentration of fermentation is 0.1%-
5%, 25-45 DEG C of fermentation temperature, fermentation time 32-48h, in 7.0-8.0, the revolving speed of shaking table concussion is the pH of fermentation medium
150-250rpm。
8. application according to claim 3, which is characterized in that in the step (2), bacillus amyloliquefaciens CFC-10 hair
Yeast-like fungi liquid is diluted with water, and strain concentration is 10 after dilution8Cfu/mL or more.
9. the application according to any one of claim 3-8, which is characterized in that in the step (2), spraying method be with
Trunk is squirted as degree, is at least sprayed 2 times, sprays interval 8-10d every time.
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| CN110079481A (en) * | 2019-05-21 | 2019-08-02 | 阜阳师范学院 | One bacillus amyloliquefaciens SL-7 and its application |
| CN111662843A (en) * | 2020-06-17 | 2020-09-15 | 中国热带农业科学院椰子研究所 | Bacillus amyloliquefaciens and separation and screening method and application thereof |
| CN114292776A (en) * | 2021-12-21 | 2022-04-08 | 湖北省生物农药工程研究中心 | Bacillus megaterium and application thereof to tea trees |
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| CN111662843A (en) * | 2020-06-17 | 2020-09-15 | 中国热带农业科学院椰子研究所 | Bacillus amyloliquefaciens and separation and screening method and application thereof |
| CN114292776A (en) * | 2021-12-21 | 2022-04-08 | 湖北省生物农药工程研究中心 | Bacillus megaterium and application thereof to tea trees |
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