CN105368747B - A strain of Bacillus amyloliquefaciens and its application - Google Patents
A strain of Bacillus amyloliquefaciens and its application Download PDFInfo
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Abstract
本发明提供了一株解淀粉芽孢杆菌菌株，所述解淀粉芽孢杆菌菌株为解淀粉芽孢杆菌IBFCBF‑1，且所述解淀粉芽孢杆菌IBFCBF‑1保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.11230。本发明提供的解淀粉芽孢杆菌菌株IBFCBF‑1具有抗真菌范围广、拮抗效果显著的特性，因而具有良好的工业化应用前景。同时，本发明提供的解淀粉芽孢杆菌菌株IBFCBF‑1对农作物具有很好的促生长效果。
The present invention provides a Bacillus amyloliquefaciens strain, the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens IBFCBF-1, and the Bacillus amyloliquefaciens IBFCBF-1 is preserved in the General Microorganism Center of China Microorganism Culture Collection Management Committee , the deposit number is CGMCC No.11230. The Bacillus amyloliquefaciens strain IBFCBF-1 provided by the invention has the characteristics of wide antifungal range and remarkable antagonistic effect, and thus has good industrial application prospect. At the same time, the Bacillus amyloliquefaciens strain IBFCBF-1 provided by the present invention has a good growth-promoting effect on crops.
Description
Technical field
The present invention relates to bacterial strain Cultivating techniques fields, more specifically, be related to a bacillus amyloliquefaciens bacterial strain and
It is applied.
Background technique
Soil-borne disease refers to that pathogen such as fungi, bacterium, nematode and virus are lived in the soil with invalid body, and condition is suitable for
When from crop root or stem infringement crop caused by disease.Soil-borne disease include stand withered, line is withered, blueness is withered, withered, epidemic disease,
It dampings off, root-rot, soft corruption, root-knot nematode, cyst nematode etc., these diseases usually infect plant root or stem, so as to cause work
Object rhizome portion or even complete stool morbidity, cause heavy economic losses.
Prevention and control, and the long-time service of chemical agent, meeting presently mainly are carried out to soil-borne disease by using chemical agent
Water source soil is caused to pollute, the ecological balance is destroyed, pesticide residue, Minor diseases are rampant and sex pheromone is made to develop drug resistance
Etc. a series of serious consequence.With the enhancing of people's environmental consciousness, the attention of food-safe problem, and to ecological environment
The requirement of bio-diversity and agricultural sustainable development is protected in construction so that biological prevention and control soil-borne disease become current research and
The hot spot of exploitation.
Bacillus (Bacillus sp.) is a kind of sporiferous gram-positive bacterium, and aerobic or amphimicrobian is raw
It is living, the endospore of heat-resisting, drought-enduring, uvioresistant and anti-organic solvent can be generated, produced gemma can be made into pulvis, wettable
The various dosage forms such as pulvis, and rear non-inactivation mixed with chemical pesticide, are ideal screening of biocontrol agents objects.In bacillus,
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) are a kind of with the active bacterium of broad-spectrum antibacterial, the Xie Dian
There is afnyloliquefaciens stronger metabolite to generate ability, can generate a variety of antibacterial substances, thus have preferable application
Prospect.
Summary of the invention
The object of the present invention is to provide a bacillus amyloliquefaciens bacterial strain and its applications, so that Xie Dian provided by the invention
Afnyloliquefaciens and its fermentation liquid produced can effectively prevent plant silborne fungal diseases.
In order to solve the above technical problem, the present invention provides following technical solutions:
The present invention provides a bacillus amyloliquefaciens bacterial strain, the Bacillus amyloliquefaciens strain is solution starch gemma
Bacillus IBFCBF-1, and the classification naming of the bacillus amyloliquefaciens IBFCBF-1 are as follows: bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens it is common that China Committee for Culture Collection of Microorganisms), and was preserved on August 11st, 2015
Microorganism center (depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；
Postcode: 100101), deposit number is CGMCC No.11230.
Preferably, the bacterium colony of the bacillus amyloliquefaciens IBFCBF-1 is creamy white, translucent, round, and edge is not whole
Together, surface wettability is smooth.
The present invention also provides the preparation method of bacillus amyloliquefaciens IBFCBF-1, the preparation method includes following step
It is rapid:
S01: soil sample is acquired by multi-point sampling method, and 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；
Pathogen fungus block: being inoculated into the center of PDA culture medium plate by S06, is being inoculated with institute away from plate center equidistant
Separation bacterium is stated, 25 DEG C of cultures select inhibition zone maximum for bacillus amyloliquefaciens IBFCBF-1.
The present invention also provides the application of bacillus amyloliquefaciens IBFCBF-1, the bacillus amyloliquefaciens IBFCBF-1
And the fermentation liquid of the bacillus amyloliquefaciens IBFCBF-1 is applied to prevention and treatment fungal disease.
Preferably, the fungal disease is that soil passes fungus diseases.
Preferably, it is Rhizoctonia solani Kuhn, flax anthrax-bacilus, sharp fusarium flax specialized form, capsicum epidemic disease that the soil, which passes fungus diseases,
Mould, tomato gray mould bacterium, Fusarium oxysporum, Fusarium graminearum, fusarium moniliforme, Rhizoctonia cerealis, early epidemic germ, gaeumannomyce
Bacterium or sclerotinite.
Preferably, the preparation of the fermentation liquid of the bacillus amyloliquefaciens IBFCBF-1 includes: by bacillus amyloliquefaciens
IBFCBF-1 is seeded in liquid fermentation medium, and in 25-30 DEG C of progress fermented and cultured, shaking table shakes 2-4 days, obtains solution starch
The fermentation liquid of bacillus IBFCBF-1.
Preferably, the revolving speed of the shaking table concussion is 180r/min.
Preferably, the liquid fermentation medium includes following components: the tryptone of 10g/L according to concentration, 5g/L's
Yeast leaches cream, the sucrose of 20g/L, the NaCl of 5g/L.
Preferably, the pH of the liquid fermentation medium is 7.2-7.4, and is sterilized 20 minutes when temperature is 121 DEG C.
The present invention provides a bacillus amyloliquefaciens bacterial strain, the Bacillus amyloliquefaciens strain is solution starch gemma
Bacillus IBFCBF-1, and to be preserved in China Committee for Culture Collection of Microorganisms general by the bacillus amyloliquefaciens IBFCBF-1
Logical microorganism center, deposit number are CGMCC No.11230.Bacillus amyloliquefaciens strain IBFCBF-1 provided by the invention
, antagonistic effect significant characteristic wide with antimycotic range, thus there is good industrial applications prospect.Meanwhile the present invention
The Bacillus amyloliquefaciens strain IBFCBF-1 of offer has good growth-promoting effect to crops.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, for those of ordinary skills, do not making the creative labor
Under the premise of, it can also be obtained according to these attached drawings other attached drawings.
Fig. 1 is the preparation flow figure of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention；
Fig. 2 is the colonial morphology figure of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention；
Fig. 3 is the Gram's staining figure of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention；
Fig. 4 is the spore staining figure of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention；
Fig. 5 is the PCR primer agarose electrophoresis figure of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention；
Fig. 6 is the systematic growth of the 16S sequence construct of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention
Tree graph；
Fig. 7 is the signal of bacillus amyloliquefaciens IBFCBF-1 antagonism flax damping-off opportunistic pathogen provided in an embodiment of the present invention
Figure；
Fig. 8 is the schematic diagram of bacillus amyloliquefaciens IBFCBF-1 prevention and treatment capsicum epidemic disease provided in an embodiment of the present invention.
Specific embodiment
Bacillus amyloliquefaciens strain provided in an embodiment of the present invention, make bacillus amyloliquefaciens provided by the invention and its
Fermentation liquid produced can effectively prevent plant silborne fungal diseases.
Technical solution in embodiment in order to enable those skilled in the art to better understand the present invention, and make of the invention real
The above objects, features, and advantages for applying example can be more obvious and easy to understand, with reference to the accompanying drawing to the technology in the embodiment of the present invention
Scheme is described in further detail.
Bacillus amyloliquefaciens strain provided in an embodiment of the present invention is bacillus amyloliquefaciens IBFCBF-1, the Xie Dian
Afnyloliquefaciens IBFCBF-1 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on August 11st, 2015
Center (abbreviation CGMCC, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute；Postcode: 100101), deposit number is CGMCC No.11230.
Attached drawing 1 is please referred to, attached drawing 1 shows the preparation of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention
Flow chart.The preparation step of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention are as follows:
S01: soil sample, and collected soil sample is finely ground, the soil sample in the embodiment of the present invention are acquired by multi-point sampling method
It is derived from Hemp Inst., China Academy of Agricultural Sciences's Yuanjiang experiment centre capsicum sample plot；
S02: finely ground soil sample being put into the centrifuge tube equipped with sterile water and is fullyd shake, and it is 10 that concentration, which is made,-1Soil
Suspension；
S03: by Soil Slurry gradient dilution step by step, the Soil Slurry of various concentration is obtained；
S04: choosing concentration is 10-5、10-6With 10-7The Soil Slurry of three gradients, and it is added to NA culture medium flat plate
Upper carry out coating process is put into 30 DEG C of incubator and cultivates 48 hours, obtains bacterium colony；
S05: selecting the different single colonie of form and carry out scribing line conservation on inclined-plane, and cultivate 24 hours, obtain separation bacterium,
The separation bacterium is placed in 4 DEG C of refrigerator and is saved backup；
S06: flax is found into withered, capsicum epidemic disease enzyme with the punch of diameter 0.5cm, cucumber fusarium axysporum opportunistic pathogen fungus block is inoculated into
The center of PDA culture medium plate, the equidistant inoculation separation bacterium at away from plate center 2.5cm right-angled intersection, while with not
The conduct of inoculation separation bacterium compares (CK), and 3 repetitions of progress per treatment are tested, and cultivates under the conditions of temperature is 25 DEG C, when right
When covering with whole vessel according to group (CK), the size of inhibition zone is recorded, selects inhibition zone maximum for bacillus amyloliquefaciens
IBFCBF-1。
Wherein, NA culture medium is nutrient agar, and the composition of the nutrient agar includes 10g/L according to concentration
Tryptone, the beef extract of 3g/L, the NaCl of 5g/L.The pH value of NA culture medium in the preparation is 7.2~7.4, and is passed through
The sterilization treatment of 20min, and temperature when sterilizing is 121 DEG C.PDA culture medium is potato dextrose agar.
The embodiment of the invention provides the schematic diagrames of bacillus amyloliquefaciens IBFCBF-1 antagonism flax damping-off opportunistic pathogen, show
Intention please refers to attached drawing 7., it can be seen that being inoculated with bacillus amyloliquefaciens when control group all covers with vessel from attached drawing 7
There is apparent inhibition zone in the PDA culture medium of IBFCBF-1 separation bacterium, and the diameter of inhibition zone is in 2.0cm or more, by
The appearance of inhibition zone can illustrate bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention to flax damping-off opportunistic pathogen
It significantly inhibits, it is thus possible to be applied to prevention and treatment fungal disease well.
The preparation of the fermentation liquid of bacillus amyloliquefaciens IBFCBF-1 provided in an embodiment of the present invention includes: that will solve starch bud
Spore bacillus IBFCBF-1 is seeded in liquid fermentation medium, and in 25-30 DEG C of progress fermented and cultured, shaking table shakes 2-4 days, obtains
The fermentation liquid of bacillus amyloliquefaciens IBFCBF-1.
Wherein, the revolving speed of shaking table concussion is 180r/min.
Liquid fermentation medium includes following components: the tryptone of 10g/L according to concentration, and the yeast of 5g/L leaches cream,
The sucrose of 20g/L, the NaCl of 5g/L.The pH value of liquid fermentation medium in the preparation is 7.2~7.4, and going out by 20min
Bacterium processing, and temperature when sterilizing is 121 DEG C.
The present invention is identified that bacterial strain provided in an embodiment of the present invention, 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.The bacterium of bacterial strain provided in an embodiment of the present invention
It falls aspect graph and please refers to attached drawing 2.
It is translucent, it can be seen that the bacterium colony of bacterial strain provided in an embodiment of the present invention is creamy white from attached drawing 2, round, side
Edge is irregular, and surface wettability is smooth.
Further, Gram's staining and spore staining carried out to bacterial strain provided in an embodiment of the present invention with kit, and
It oily microscopic observation bacterial strain and takes pictures to bacterial strain.The Gram's staining of the bacterial strain and spore staining please refer to attached drawing 3 and attached drawing
4。
From attached drawing 3, it can be seen that after Gram's staining, bacterial strain provided in an embodiment of the present invention is rod-shaped, and in indigo plant
Purple is gram-positive bacteria.From attached drawing 4, it can be seen that after spore staining, bacterial strain thallus provided in an embodiment of the present invention
Aobvious blue, the aobvious red of gemma, thus illustrates that bacterial strain provided by the invention can generate gemma.
2, Physiology and biochemistry is identified
(1) catalase is tested
3% hydrogen peroxide is directly added dropwise in the liquid medium of bacterial strain, observes immediately.If there is a large amount of bubbles to generate,
It is then the positive；If not generating bubble, for feminine gender.Bacterial strain provided by the invention generates a large amount of bubbles immediately, and experimental result is sun
Property.
(2) oxidizing ferment is tested
One jiao of extracting waste cleaning filter paper dips a small amount of bacterial strain bacterium colony, and the hydrochloride base that concentration is 1% is added to benzene two
Amine aqueous solution one drips, positive pinkiness immediately, and color can gradually deepen.In this experiment, bacterium colony pinkiness, face
Color is gradually deepened, and experimental result is the positive.
(3) Starch Hydrolysis is tested
The strain point of bacterial strain is connected on starch culture-medium, is cultivated for 24 hours under conditions of temperature is 37 DEG C, a small amount of iodine is added dropwise
Liquid gently rotates on starch culture-medium plate, is evenly distributed on iodine solution on starch culture-medium plate, observes periphery of bacterial colonies
Situation.If colorless and transparent circle, which occurs, in periphery of bacterial colonies shows the ability for having hydrolysis starch, conversely, not having.The week of this bacterial strain bacterium colony
It is with transparent circle generation, the ability that there is hydrolysis starch thus, it is possible to illustrate this bacterial strain.
(4) methyl red MR is tested
This small amount of bacterial strain of picking, and be inoculated on collective media, it is cultivated 3~5 days under conditions of temperature is 30 DEG C,
Culture solution 1ml is taken after culture, and the drop of methyl red indicator 1~2 is added, and the positive is in cerise, and weakly positive is in pale red, yin
Property is yellow.In this experiment, bacterium solution turns yellow, so for feminine gender.
(5) VP is tested
This small amount of bacterial strain of picking, and it is inoculated in collective media, it is cultivated 4 days under conditions of temperature is 30 DEG C, culture
After take culture solution 2.5ml that a naphthols absolute alcohol solution 0.6ml is first added, add concentration be 40% potassium hydroxide it is water-soluble
Liquid 0.2ml shakes 2-5min, and red is usually presented in positive bacteria immediately, if redfree occurs, is statically placed in the constant temperature of room temperature or 30 DEG C
In case, if not showing red in 2h still, it can determine that as feminine gender.In this experiment, bacterium solution reddens immediately, so for the positive.
(6) gelatin liquefaction is tested
This bacterial strain is taken, percutaneous puncture-inoculation is located at this bacterial strain at the 2/3 of gelatin depth in gelatin.It is 20 DEG C in temperature
Under conditions of cultivate 5-7d.Whether observation this bacterial strain of result is liquefied by bacterium daily, positive for test if being liquefied；If no
It is liquefied, is then feminine gender.In this experiment, gelatin is liquefied, therefore this bacterial strain is the positive.
(7) nitrate reduction is tested
By this strain inoculated in nitrate broth, then shaking table culture 3d under conditions of temperature is 28 DEG C takes 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,
Conversely, not changing color as feminine gender.In this experiment, bacterium solution turns yellow, and illustrates this bacterial strain for the positive.
(8) hydrogen sulfide experiment is produced
By this bacterial strain percutaneous puncture-inoculation in lead acetate medium, 24~48h is cultivated under conditions of temperature is 35 DEG C, and see
Examine result.If culture medium blackening, for the positive；Not blackening is then feminine gender.In this experiment, culture medium does not change colour, illustrates this
Bacterial strain is feminine gender.
(9) citrate utilizes experiment
The streak inoculation on simon Si Shi citrate medium inclined-plane of this bacterial strain is chosen, is trained under conditions of temperature is 37 DEG C
It supports 3-7 days.If culture medium is alkalinity person, i.e., indicator blue or pink are the positive；If culture medium is non-discolouring, for yin
Property.In this experiment, culture medium does not change colour, illustrates this bacterial strain for feminine gender.
(10) lecithin activity is tested
The surface of Fresh Egg is carried out disinfection with 75% ethyl alcohol, and egg is beaten into a hole with the tweezers of sterilizing, is inclined
Then yolk is sucked out with aseptic straw in egg white, be added in the NA culture medium for being cooled to 50 DEG C or so after melting, after being mixed evenly
Fell plate, and point connects this bacterial strain, cultivates for 24 hours under conditions of temperature is 30 DEG C, and observed.If colony edge occurs muddy
Circle person is enzyme positive.In this experiment, there is apparent muddy circle in colony edge, illustrates this bacterial strain for the positive.
(11) malonate utilizes experiment
Picking culture 12h lawn is inoculated in malonate culture medium, cultivates 24-48h under conditions of temperature is 35 DEG C, trains
Base is supported by green change indigo plant person as the positive, otherwise is feminine gender.In this experiment, culture medium does not change colour, illustrates this bacterial strain for feminine gender.
(12) glucose fermentation is tested
A small amount of this bacterial strain percutaneous puncture-inoculation of picking is cultivated under conditions of temperature is 30 DEG C in glucose oxidation-fermentation medium
3d observes the variation of culture medium color.If needing to continue to observe 7d without color change, culture medium flavescence person is fermented type.?
This time in experiment, culture medium turns yellow, and illustrates this bacterial strain for the positive.
(13) cellulose decomposition is tested
When having obvious bacterium colony, a drop is instilled into plate on sodium carboxymethylcellulose solid medium for spread plate
Congo red dye liquor is managed, and is evenly distributed in Congo red dye liquor on plate.1ml sodium chloride solution is added after 15 minutes, impregnates
After 15 minutes, dyeing is washed away, sees whether to generate transparent circle.If there is transparent circle generation, for the positive.In this experiment, have
Transparent circle generates, and illustrates this bacterial strain for the positive.
(14) galactose utilization experiment is positive
By this strain inoculated in gala sugar culture-medium, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use galactolipin if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth illustrates that this bacterial strain can
To utilize galactolipin.
(15) arabinose utilizes experiment
By this strain inoculated in arabinose culture medium, 2d is cultivated under conditions of temperature is 30 DEG C, observation bacterium colony is raw
Long situation can use arabinose if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth illustrates this bacterium
Strain can use arabinose.
(16) mannose utilizes experiment
By this strain inoculated in sweet dew sugar culture-medium, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use mannose if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth, this bacterial strain can benefit
Use mannose.
(17) D-Fructose utilizes experiment
By this strain inoculated in D-Fructose culture medium, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use D-Fructose if bacterium colony is formed, conversely, not all right.In this experiment, thalli growth, this bacterial strain can benefit
Use D-Fructose.
(18) D- xylose utilization is tested
By this strain inoculated in D- xylose media, 2d, observation bacterium colony growth are cultivated under conditions of temperature is 30 DEG C
Situation can use D- xylose if bacterium colony is formed, conversely, not all right.In this experiment, thallus is not grown, this bacterial strain can not
To utilize D- xylose.
To sum up, Physiology and biochemistry qualification result such as table 1.
Table 1: the Physiology and biochemistry qualification result of this bacterial strain
| 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 | - |
Wherein ,+be expressed as this bacterial strain and have reaction or can use ,-be expressed as this bacterial strain and do not react or cannot utilize.
3,16S rDNA sequence is analyzed
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 H of 18 μ L2O；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 extend 1min；30 circulations, 72 DEG C of extension 5min, 4 DEG C of preservations.
Attached drawing 5 is please referred to, the PCR product that attached drawing 5 shows bacillus IBFCBF-1 provided in an embodiment of the present invention is used
The electrophoretogram of 1% agarose gel electrophoresis detection.PCR product is sequenced by Changsha your Biotechnology Co., Ltd of dimension generation, is surveyed
Sequence result is referring to the nucleotide sequence in subordinate list.Gained sequence is subjected to homology sequence by NCBI-BLAST and compares analysis,
Obtain the higher sequence of similitude.With MEGA6.06 software building phylogenetic tree, the 16S rDNA sequence and solution of this bacterial strain
The homology of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) reaches 100%, and the development tree graph of this bacterial strain is asked
With reference to attached drawing 6.
In summary morphological observation, Physiology and biochemistry identification and 16S rDNA the sequencing results, can determine this bacterial strain
IBFCBF-1 is bacillus amyloliquefaciens section bacillus (Bacillus amyloliquefaciens), is named as Xie Dian
Afnyloliquefaciens IBFCBF-1.
Bacillus amyloliquefaciens IBFCBF-1 and its fermentation liquid provided in an embodiment of the present invention can be applied to prevention and treatment fungi
Disease, the fungal disease are that soil biography fungus diseases are Rhizoctonia solani Kuhn (Rhizoctonia solani), flax anthrax-bacilus
(Colletotrichum linicola), sharp fusarium flax specialized form (Fusarium oxysporum Schltdl.ex
Snyder et Hansen f.sp.lini), phytophthora blight of pepper (Phytophthora capsici), tomato gray mould bacterium
(Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), Fusarium graminearum (F.graminearum),
Fusarium moniliforme (F.verticillioides), Rhizoctonia cerealis (R.cerealis), early epidemic germ (Alternaria
Solani), gaeumannomyce bacterium (Gaeumannomyces graminis) or sclerotinite (Sclerotinia sclerotiorum)
Deng soil pass fungus diseases.
The embodiment of the present invention has carried out bacillus amyloliquefaciens IBFCBF-1 and its fermentation liquid pair by taking phytophthora blight of pepper as an example
The research of fungal disease, research contents are as follows.
(1) selection of culture medium
The culture of bacillus amyloliquefaciens IBFCBF-1 uses NB fluid nutrient medium (i.e. beef extract-peptone Liquid Culture
Base), the preparation of the NB fluid nutrient medium includes the peptone of 10g, the beef extract of 3g and the NaCl of 5g, above-mentioned substance use go from
Sub- water is settled to 1000ml, and adjusting pH value is 7.2-7.4, then 1 × 105Sterilize 20min under the pressure of Pa, and NB liquid is made
Body culture medium.
Capsicum epidemic disease enzymophathy fungal pathogens use PDA liquid medium, and the preparation of the PDA liquid medium includes the peeling of 200g
Potato, the glucose of 20g, above-mentioned substance are settled to 1000mL using deionized water, and adjusting pH value is 7.2-7.4, then
1 × 105Sterilize 20min under the pressure of Pa, and PDA liquid medium is made.
(2) selection of capsicum variety
The Sweet Pepper Varieties eggplant door that capsicum selects Vegetable Research Inst., Hunan Prov. Agriculture Science Academy to provide.By the seed of eggplant door with dense
Degree carries out surface sterilization 20min for 10% hydrogen peroxide, then uses sterile water shower 3 times, and be placed inCulture dish
In, constant temperature half-light vernalization under conditions of temperature is 30 DEG C.The seed for choosing a length of 0.5cm of bud is sowed in 9 hole alms bowls, and matrix is city
Nutrition Soil is sold, capsicum is placed in 30 ± 1 DEG C of growth cabinet and is cultivated, and the intensity of illumination at this time is 12000Lux, illumination
Period is L ︰ D=14 ︰ 10, RH=80% ± 10%.
(3) specific experiment designs
Experiment is divided into 3 groups:
CK1: clear water；CK2:NB culture medium；T: bacillus amyloliquefaciens IBFCBF-1 bacterium solution.
Wherein, bacillus amyloliquefaciens IBFCBF-1 is 30 DEG C in temperature, and revolving speed is cultivated under conditions of being 180r/min
Then bacterium solution is diluted to 10 with sterile water by 48h5CFU/mL.Capsicum elicitin pathogenic bacteria is 30 DEG C in temperature, revolving speed 180r/
7d is cultivated under conditions of min, and bacterium solution is then diluted to 10 with sterile water5CFU/mL。
In experiment, pepper seed two of every hole sowing budding in 9 hole alms bowls guarantee 18 plants of young plant survivals, and by 18 plants
Young plant is divided into three groups, when capsicum was cultivated to the 3-4 leaf phase, applies capsicum elicitin pathogenic bacteria bacterium solution 2.5mL in the rhizosphere of capsicum, so
It is divided into the clear water, NB culture medium and bacillus amyloliquefaciens IBFCBF-1 bacterium solution for adding 2.5mL in three groups of young plant respectively backward.
Apply 5mL nutrient solution every the every hole 2d, to guarantee that matrix is more wet, capsicum being capable of normal growth.Chili growth situation is observed,
Plant height, stem thickness, internode are carried out away from, top half fresh weight and dry weight and the growth indexes such as root system fresh weight and dry weight to capsicum after 40d
Measurement, count every group of capsicum epidemic disease morbidity strain number.
(4) measurement method
From pepper plant base portion to stem, the distance between top trunk diameter growth point is plant height, the nearly root knot of plant when measurement
First segment stem diameter be stem thickness.The length of the every trifle stem of plant is internode away from the first-half of nearly root knot is chosen in this experiment.It will
Pepper plant is cut from first node of nearly root knot, is weighed the weight of top half and root system part respectively, is denoted as fresh weight, so
It is placed into 85 DEG C of baking oven that drying to constant weight afterwards, weighs its weight respectively again, be denoted as their dry weight.Experimental result please join
Examine table 2, table 3 and attached drawing 8.
Influence of 2: the three kinds of different disposal methods of table to chili growth
Table 3: a situation arises for the capsicum epidemic disease handled with three kinds of distinct methods
| Processing | It falls ill strain number (strain) | Disease incidence (%) | Control efficiency (%) |
| T | 2.5±0.25c | 13.89±1.38c | 77.27±2.25 |
| CK2 | 9.5±0.31b | 52.78±1.72b | 13.63±2.81 |
| CK1 | 11.0±0.38a | 61.11±2.11a | - |
Wherein, diseased plant rate %=morbidity strain number/investigation total strain number × 100；Control efficiency %=(1- processing group morbidity strain
Rate/control group diseased plant rate) × 100.
From table 2, it can be seen that the processing of T group make capsicum have higher plant height, thicker stem, and first segment pitch,
Second successively away from, top half fresh weight, top half dry weight, root fresh weight and root dry weight data are all than remaining two groups data
Greatly.Meanwhile it can be seen that pepper plant grows fine from attached drawing 8, thus, solution starch gemma bar provided in an embodiment of the present invention
Bacterium IBFCBF-1 and its fermentation liquid can effectively promote the growth of plant., it can be seen that the processing of T group makes capsicum from table 3
Disease incidence be significantly lower than other two groups of disease incidence, and control efficiency be even more be significantly greater than other two groups of control efficiency, by
This can illustrate that bacillus amyloliquefaciens IBFCBF-1 and its fermentation liquid provided in an embodiment of the present invention are imitated with significant antagonism
Fruit.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within made modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Claims (6)
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| CN108148778B (en) * | 2018-01-10 | 2021-08-20 | 江苏耕耘化学有限公司 | Bacillus amyloliquefaciens GY30 and preparation and application of bacterial powder thereof |
| CN109182197B (en) * | 2018-09-29 | 2021-04-27 | 淮阴工学院 | A kind of Bacillus amyloliquefaciens CPLK1314 and its application in feed storage |
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