CN105368747A - Bacillus amyloliquefaciens strain and application thereof - Google Patents
Bacillus amyloliquefaciens strain and application thereof Download PDFInfo
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Abstract
The invention provides a Bacillus amyloliquefaciens strain which is Bacillus amyloliquefaciens IBFCBF-1. The Bacillus amyloliquefaciens IBFCBF-1 is collected in the China General Microbiological Culture Collection Center and numbered as CGMCC No.11230. The Bacillus amyloliquefaciens IBFCBF-1 has the characteristics of wide antifungal range and remarkable antagonistic effect, thereby having a good industrialization application prospect. In addition, the Bacillus amyloliquefaciens IBFCBF-1 has good effect on promoting growth of crops.
Description
Technical field
The present invention relates to bacterial strain Cultivating techniques field, more specifically, relate to bacillus amyloliquefaciens bacterial strain and an application thereof.
Background technology
Soil-borne disease refers to that pathogenic agent such as fungi, bacterium, nematode and virus are lived in soil with invalid body, the disease caused from crop root or stem infringement crop when condition is suitable for.Soil-borne disease comprise vertical withered, line is withered, green grass or young crops is withered, withered, epidemic disease, damping off, root-rot, soft corruption, root knot nematode, Cyst nematode etc., these diseases infect plant root or stem usually, thus cause root of the crop stem and even complete stool morbidity, cause heavy economic losses.
At present mainly by adopting chemical agent to carry out prevention and control to soil-borne disease, and the life-time service of chemical agent, water source soil pollution can be caused, the eubiosis is destroyed, pesticide residue, Minor diseases are rampant and make a series of serious consequences such as sex pheromone develops immunity to drugs.Along with the enhancing of people's environmental consciousness, to the attention of food-safety problem, and to improvement of the ecological environment, the requirement of protection species diversity and agricultural sustainable development, makes biological prevention and control soil-borne disease become the focus of current research and exploitation.
Genus bacillus (Bacillussp.) is the sporiferous gram positive bacterium of a class, aerobic or amphimicrobian is lived, the statospore of heat-resisting, drought-enduring, uvioresistant and anti-organic solvent can be produced, produce gemma and can be made into the various formulation such as pulvis, wettable powder, and rear non-inactivation mixed with chemical pesticide, be desirable screening of biocontrol agents object.In genus bacillus, bacillus amyloliquefaciens (Bacillusamyloliquefaciens) is a kind of bacterium with broad-spectrum antibacterial activity, this bacillus amyloliquefaciens has stronger meta-bolites and produces ability, multiple antibacterial substance can be produced, thus there is good application prospect.
Summary of the invention
The object of this invention is to provide bacillus amyloliquefaciens bacterial strain and an application thereof, effectively prevent and treat plant silborne fungal diseases to enable bacillus amyloliquefaciens provided by the invention and the fermented liquid produced thereof.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
The invention provides a bacillus amyloliquefaciens bacterial strain, described Bacillus amyloliquefaciens strain is bacillus amyloliquefaciens IBFCBF-1, and the Classification And Nomenclature of described bacillus amyloliquefaciens IBFCBF-1 is: bacillus amyloliquefaciens (Bacillusamyloliquefaciens), and be preserved in (depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 11st, 2015; Postcode: 100101), deposit number is CGMCCNo.11230.
Preferably, the bacterium colony of described bacillus amyloliquefaciens IBFCBF-1 is creamy white, translucent, and circular, edge is irregular, and surface wettability is smooth.
Present invention also offers the preparation method of bacillus amyloliquefaciens IBFCBF-1, described preparation method comprises the following steps:
S01: gather soil sample by multi-point sampling method, and the soil sample porphyrize that will collect;
S02: the soil sample of porphyrize is put into the centrifuge tube that sterilized water is housed and fully shakes, obtain Soil Slurry;
S03: by described Soil Slurry gradient dilution, obtain the Soil Slurry of different concns;
S04: choosing concentration is 10
-5, 10
-6with 10
-7soil Slurry, and join and NA culture medium flat plate carry out coating process, be put in the incubator of 30 DEG C and cultivate, obtain bacterium colony;
S05: select single bacterium colony that form differs and carry out line conservation, cultivate 24 hours, obtain isolate, the refrigerator that described isolate is placed in 4 DEG C is saved backup;
S06: center pathogenic bacteria bacterium block being inoculated into PDA culture medium flat plate, inoculating described isolate, 25 DEG C of cultivations apart from plate center equidistant, that selection inhibition zone is maximum is bacillus amyloliquefaciens IBFCBF-1.
Present invention also offers the application of bacillus amyloliquefaciens IBFCBF-1, the fermented liquid of described bacillus amyloliquefaciens IBFCBF-1 and described bacillus amyloliquefaciens IBFCBF-1 is applied to control fungal disease.
Preferably, described fungal disease is that soil passes fungus diseases.
Preferably, described soil passes fungus diseases is dry thread Pyrenomycetes, flax anthrax-bacilus, sharp sickle spore flax specialized form, phytophthora blight of pepper, tomato gray mould bacterium, Fusarium oxysporum, Fusarium graminearum, fusarium moniliforme, Rhizoctonia cerealis, early epidemic germ, gaeumannomyce bacterium or sclerotinite.
Preferably, the preparation of the fermented liquid of described bacillus amyloliquefaciens IBFCBF-1 comprises: be seeded in liquid fermentation medium by bacillus amyloliquefaciens IBFCBF-1, carry out fermentation culture at 25-30 DEG C, shaking table concussion 2-4 days, obtains the fermented liquid of bacillus amyloliquefaciens IBFCBF-1.
Preferably, the rotating speed of described shaking table concussion is 180r/min.
Preferably, described liquid fermentation medium comprises following component according to concentration: the Tryptones of 10g/L, and the yeast of 5g/L leaches cream, the sucrose of 20g/L, the NaCl of 5g/L.
Preferably, the pH of described liquid fermentation medium is 7.2-7.4, and the sterilizing 20 minutes when temperature is 121 DEG C.
The invention provides a bacillus amyloliquefaciens bacterial strain, described Bacillus amyloliquefaciens strain is bacillus amyloliquefaciens IBFCBF-1, and described bacillus amyloliquefaciens IBFCBF-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.11230.Bacillus amyloliquefaciens strain IBFCBF-1 provided by the invention has that antimycotic scope is wide, the significant characteristic of antagonistic effect, thus has good industrial applications prospect.Meanwhile, Bacillus amyloliquefaciens strain IBFCBF-1 provided by the invention has good growth-promoting effect to farm crop.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, for those of ordinary skills, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the preparation flow figure of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides;
Fig. 2 is the colonial morphology figure of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides;
Fig. 3 is the gramstaining figure of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides;
Fig. 4 is the spore staining figure of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides;
Fig. 5 is the PCR primer agarose electrophoresis figure of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides;
Fig. 6 is the phylogeny tree graph of the 16S sequence construct of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides;
Fig. 7 is the schematic diagram of the former bacterium of bacillus amyloliquefaciens IBFCBF-1 antagonism flax damping-off that the embodiment of the present invention provides;
Fig. 8 is the schematic diagram of the bacillus amyloliquefaciens IBFCBF-1 control capsicum epidemic disease that the embodiment of the present invention provides.
Embodiment
The Bacillus amyloliquefaciens strain that the embodiment of the present invention provides, enables bacillus amyloliquefaciens provided by the invention and the fermented liquid produced thereof effectively prevent and treat plant silborne fungal diseases.
Technical scheme in the embodiment of the present invention is understood better in order to make those skilled in the art person, and enable the above-mentioned purpose of the embodiment of the present invention, feature and advantage become apparent more, below in conjunction with accompanying drawing, the technical scheme in the embodiment of the present invention is described in further detail.
The Bacillus amyloliquefaciens strain that the embodiment of the present invention provides is bacillus amyloliquefaciens IBFCBF-1, described bacillus amyloliquefaciens IBFCBF-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 11st, 2015 and (is called for short CGMCC, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Postcode: 100101), deposit number is CGMCCNo.11230.
Please refer to accompanying drawing 1, figure 1 show the preparation flow figure of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides.The preparation process of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides is:
S01: gather soil sample by multi-point sampling method, and the soil sample porphyrize that will collect, the soil sample in the embodiment of the present invention takes from Hemp Inst., China Academy of Agricultural Sciences's Yuanjiang experiment centre capsicum sample plot;
S02: the soil sample of porphyrize is put into the centrifuge tube that sterilized water is housed and fully shakes, making concentration is 10
-1soil Slurry;
S03: by described Soil Slurry gradient dilution step by step, obtain the Soil Slurry of different concns;
S04: choosing concentration is 10
-5, 10
-6with 10
-7the Soil Slurry of three gradients, and join and NA culture medium flat plate carries out coating process, be put in the incubator of 30 DEG C and cultivate 48 hours, obtain bacterium colony;
S05: select single bacterium colony that form differs and carry out line conservation on inclined-plane, and cultivate 24 hours, obtain isolate, the refrigerator that described isolate is placed in 4 DEG C is saved backup;
S06: flax is stood withered, that capsicum epidemic disease enzyme, cucumber fusarium axysporum former bacterium bacterium block are inoculated into PDA culture medium flat plate center with the punch tool of diameter 0.5cm, at the described isolate of the distance plate center equidistant inoculation of 2.5cm right-angled intersection place, simultaneously not inoculate (CK) in contrast of isolate, each process is carried out repeating experiment for 3 times, cultivate under temperature is 25 DEG C of conditions, when control group (CK) covers with whole vessel, the size of record inhibition zone, that selection inhibition zone is maximum is bacillus amyloliquefaciens IBFCBF-1.
Wherein, NA substratum is nutrient agar, and the composition of this nutrient agar comprises the Tryptones of 10g/L, the extractum carnis of 3g/L according to concentration, the NaCl of 5g/L.NA substratum pH value is in the preparation 7.2 ~ 7.4, and through the sterilising treatment of 20min, and temperature during sterilizing is 121 DEG C.PDA substratum is potato dextrose agar.
Embodiments provide the schematic diagram of the former bacterium of bacillus amyloliquefaciens IBFCBF-1 antagonism flax damping-off, schematic diagram please refer to accompanying drawing 7.Can find out from accompanying drawing 7, when control group all covers with vessel, inoculate to have on the PDA substratum of bacillus amyloliquefaciens IBFCBF-1 isolate and occurred obvious inhibition zone, and the diameter of inhibition zone is all at more than 2.0cm, can illustrate that the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides has obvious restraining effect to the former bacterium of flax damping-off by the appearance of inhibition zone, thus can be good at being applied to control fungal disease.
The preparation of the fermented liquid of the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides comprises: be seeded in liquid fermentation medium by bacillus amyloliquefaciens IBFCBF-1, fermentation culture is carried out at 25-30 DEG C, shaking table concussion 2-4 days, obtains the fermented liquid of bacillus amyloliquefaciens IBFCBF-1.
Wherein, the rotating speed of shaking table concussion is 180r/min.
Liquid fermentation medium comprises following component according to concentration: the Tryptones of 10g/L, and the yeast of 5g/L leaches cream, the sucrose of 20g/L, the NaCl of 5g/L.Liquid fermentation medium pH value is in the preparation 7.2 ~ 7.4, and through the sterilising treatment of 20min, and temperature during sterilizing is 121 DEG C.
The present invention identifies the bacterial strain that the embodiment of the present invention provides, and this qualification comprises following content:
1, Morphological Identification
The bacterial strain embodiment of the present invention provided is scoring on NA culture medium flat plate, and then being reversed by flat board, is cultivate 24h under the condition of 30 DEG C in temperature, observes and records the dull and stereotyped growing state going up bacterium colony.The colonial morphology figure of the bacterial strain that the embodiment of the present invention provides please refer to accompanying drawing 2.
Can find out from accompanying drawing 2, the bacterium colony of the bacterial strain that the embodiment of the present invention provides is creamy white, translucent, and circular, edge is irregular, and surface wettability is smooth.
Further, with test kit, gramstaining and spore staining are carried out to the bacterial strain that the embodiment of the present invention provides, and at oily Microscopic observation bacterial strain with take pictures to bacterial strain.The gramstaining of this bacterial strain and spore staining please refer to accompanying drawing 3 and accompanying drawing 4.
Can find out from accompanying drawing 3, after gramstaining, the bacterial strain that the embodiment of the present invention provides is shaft-like, and in bluish voilet, is gram-positive microorganism.Can find out from accompanying drawing 4, after spore staining, the bacterial strain thalline that the embodiment of the present invention provides is aobvious blue, and gemma is aobvious red, illustrates that bacterial strain provided by the invention can produce gemma thus.
2, Physiology and biochemistry qualification
(1) catalase experiment
In the liquid medium of bacterial strain, directly drip the hydrogen peroxide of 3%, observe immediately.If there is a large amount of bubble to produce, be then positive; If do not produce bubble, be then negative.Bacterial strain provided by the invention produces a large amount of bubble immediately, and experimental result is positive.
(2) oxydase experiment
The clean filter paper one jiao of extracting waste, dip a small amount of bacterial strain bacterium colony, add the hydrochloride base Ursol D aqueous solution one that concentration is 1%, positive is pinkiness immediately, and color can be deepened gradually.In this experiment, bacterium colony pinkiness, color is deepened gradually, and experimental result is positive.
(3) Starch Hydrolysis experiment
The bacterial classification of bacterial strain point is received on starch culture-medium, is cultivate 24h under the condition of 37 DEG C in temperature, drips a small amount of iodine liquid on starch culture-medium flat board, rotate gently, make iodine liquid be evenly distributed on starch culture-medium flat board, observe the situation of periphery of bacterial colonies.If periphery of bacterial colonies occurs that water white transparency circle shows to have amylatic ability, otherwise, then do not have.The surrounding of this bacterial strain bacterium colony has transparent circle to produce, and can illustrate that this bacterial strain has amylatic ability thus.
(4) methyl red MR tests
This bacterial strain that picking is a small amount of, and be inoculated on collective media, be cultivate 3 ~ 5 days under the condition of 30 DEG C in temperature, cultivate after terminating and get nutrient solution 1ml, and add methyl red indicator 1 ~ 2, positive in bright red, the weak positive is incarnadine, and feminine gender is yellow.In this experiment, bacterium liquid turns yellow, and institute thinks feminine gender.
(5) VP experiment
This bacterial strain that picking is a small amount of, and be inoculated in collective media, be cultivate 4 days under the condition of 30 DEG C in temperature, get nutrient solution 2.5ml after cultivation terminates and first add a naphthols straight alcohol solution 0.6ml, then add the potassium hydroxide aqueous solution 0.2ml that concentration is 40%, shake 2-5min, positive bacteria presents redness usually immediately, if redfree occurs, is statically placed in the thermostat container of room temperature or 30 DEG C, if still do not manifest redness in 2h, then feminine gender can be judged to be.In this experiment, bacterium liquid reddens immediately, and institute thinks the positive.
(6) gelatine liquefication experiment
Get this bacterial strain, percutaneous puncture-inoculation in gelatin, and makes this bacterial strain be positioned at 2/3 place of the gelatin degree of depth.Be cultivate 5-7d under the condition of 20 DEG C in temperature.Every day, whether this bacterial strain of observations was liquefied by bacterium, if be liquefied, then for test is positive; If be not liquefied, be then negative.In this experiment, gelatin is liquefied, and therefore this bacterial strain is positive.
(7) nitrate reduction experiment
By this inoculation in nitrate broth, be that under the condition of 28 DEG C, 3d cultivated by shaking table in temperature, then get 5mL nutrient solution, illustrate by test kit (Hai Bo Bioisystech Co., Ltd nitrate reduction test kit) and add developer, turn yellow as positive, otherwise, do not change color as feminine gender.In this experiment, bacterium liquid turns yellow, and illustrates that this bacterial strain is for positive.
(8) hydrogen sulfide experiment is produced
By this bacterial strain percutaneous puncture-inoculation in lead acetate medium, be cultivate 24 ~ 48h under the condition of 35 DEG C in temperature, and observations.If substratum blackening, be then positive; Not blackening is then negative.In this experiment, the non-variable color of substratum, illustrates that this bacterial strain is for negative.
(9) Citrate trianion utilizes experiment
Choosing the streak inoculation on Xi Mengsishi citrate medium inclined-plane of this bacterial strain, is cultivate 3-7 days under the condition of 37 DEG C in temperature.If substratum is alkaline person, namely indicator is blue or pinkish is positive; If substratum nondiscoloration, be then negative.In this experiment, the non-variable color of substratum, illustrates that this bacterial strain is for negative.
(10) lecithin activity experiment
The surface of Fresh Egg carries out disinfection by the ethanol with 75%, and with the tweezers of sterilizing, egg is beaten a hole, incline egg white, then aseptic straw sucking-off yolk is used, be cooled in the NA substratum of about 50 DEG C after joining thawing, fell after being mixed evenly flat board, and point connects this bacterial strain, be cultivate 24h under the condition of 30 DEG C in temperature, and observe.If colony edge occurs that muddy circle person is for enzyme positive.In this experiment, there is significantly muddy circle in colony edge, illustrates that this bacterial strain is for positive.
(11) malonate utilizes experiment
Picking is cultivated 12h lawn and is inoculated in malonate substratum, is cultivate 24-48h under the condition of 35 DEG C in temperature, and substratum is the positive by the blue person of green change, otherwise is negative.In this experiment, the non-variable color of substratum, illustrates that this bacterial strain is for negative.
(12) glucose fermentation experiment
A small amount of this bacterial strain percutaneous puncture-inoculation of picking, in glucose oxidase fermention medium, is cultivate 3d under the condition of 30 DEG C in temperature, observes the change of substratum color.If without colour-change, then need to continue to observe 7d, substratum flavescence person is fermented type.In this experiment, substratum turns yellow, and illustrates that this bacterial strain is for positive.
(13) cellulose decomposition experiment
Spread plate, on Xylo-Mucine solid medium, in time having obvious bacterium colony, instills the Congo red dye liquor of a dropper, and Congo red dye liquor is evenly distributed on flat board in flat board.Add 1ml sodium chloride solution after 15 minutes, soak after 15 minutes, wash away dyeing, observe whether produce transparent circle.If there is transparent circle to produce, be then positive.In this experiment, there is transparent circle to produce, illustrate that this bacterial strain is for positive.
(14) galactose utilization experiment is positive
By this inoculation in semi-lactosi substratum, be cultivate 2d under the condition of 30 DEG C in temperature, observe colony growth situation, if bacterium colony is formed, then can utilize semi-lactosi, otherwise, not all right.In this experiment, thalli growth, illustrates that this bacterial strain can utilize semi-lactosi.
(15) pectinose utilizes experiment
By this inoculation in pectinose substratum, be cultivate 2d under the condition of 30 DEG C in temperature, observe colony growth situation, if bacterium colony is formed, then can utilize pectinose, otherwise, not all right.In this experiment, thalli growth, illustrates that this bacterial strain can utilize pectinose.
(16) seminose utilizes experiment
By this inoculation in seminose substratum, be cultivate 2d under the condition of 30 DEG C in temperature, observe colony growth situation, if bacterium colony is formed, then can utilize seminose, otherwise, not all right.In this experiment, thalli growth, this bacterial strain can utilize seminose.
(17) D-Fructose utilizes experiment
By this inoculation in D-Fructose substratum, be cultivate 2d under the condition of 30 DEG C in temperature, observe colony growth situation, if bacterium colony is formed, then can utilize D-Fructose, otherwise, not all right.In this experiment, thalli growth, this bacterial strain can utilize D-Fructose.
(18) D-xylose utilization experiment
By this inoculation in D-xylose media, be cultivate 2d under the condition of 30 DEG C in temperature, observe colony growth situation, if bacterium colony is formed, then can utilize D-wood sugar, otherwise, not all right.In this experiment, thalline does not grow, and this bacterial strain cannot utilize D-wood sugar.
To sum up, Physiology and biochemistry qualification result is as table 1.
Table 1: the Physiology and biochemistry qualification result of this bacterial strain
| Table property feature | Response feature | Table property feature | Response feature |
| Catalase measures | + | Citrate trianion utilizes | - |
| Oxidase assay | + | Lecithin activity measures | + |
| Starch Hydrolysis measures | + | Malonate utilizes | - |
| Methyl red MR measures | - | Cellulose decomposition | + |
| VP tests | + | Galactose utilization | + |
| Gelatine liquefication measures | + | Pectinose utilizes | + |
| Nitrate reduction measures | + | Seminose utilizes | + |
| Glucose fermentation | + | D-Fructose utilizes | + |
| Product hydrogen sulfide measures | - | D-xylose utilization | - |
Wherein ,+be expressed as this bacterial strain to respond and maybe can utilize ,-being expressed as this bacterial strain does not react and maybe cannot utilize.
3,16SrDNA sequential analysis
The embodiment of the present invention adopts health to be the DNA that century bio tech ltd's genome extraction test kit extracts in this bacterial strain.
Wherein, said extracted test kit comprises the PCR reaction system of 50 μ L, and PCR reaction system comprises the 2 × MasterMix of 25 μ L; The upstream primer of 2.5 μ L; The downstream primer of 2.5 μ L; The ddH of 18 μ L
2o; The template DNA of 2 μ L.
PCR reaction conditions is: be 94 DEG C of denaturation 5min in temperature; 94 DEG C of sex change 0.5min; 53 DEG C of annealing 0.5min; 72 DEG C extend 1min; 30 circulations, 72 DEG C extend 5min, 4 DEG C of preservations.
Please refer to accompanying drawing 5, the electrophorogram that the PCR primer fig. 5 illustrating the genus bacillus IBFCBF-1 that the embodiment of the present invention provides detects with 1% agarose gel electrophoresis.PCR primer is tieed up your bio tech ltd of generation by Changsha and is checked order, and sequencing result is see the nucleotide sequence in subordinate list.Gained sequence is carried out homology sequence compare of analysis by NCBI-BLAST, obtains the sequence that similarity is higher.Use MEGA6.06 software building phylogenetic tree, the homology of the 16SrDNA sequence of this bacterial strain and bacillus amyloliquefaciens (Bacillusamyloliquefaciens) reaches 100%, and the growth tree graph of this bacterial strain please refer to accompanying drawing 6.
The qualification of comprehensive above-mentioned morphological observation, Physiology and biochemistry and 16SrDNA the sequencing results, can determine that this bacterial strain IBFCBF-1 is bacillus amyloliquefaciens section genus bacillus (Bacillusamyloliquefaciens), it is named as bacillus amyloliquefaciens IBFCBF-1.
The bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides and fermented liquid thereof can be applied to control fungal disease, this fungal disease is dry thread Pyrenomycetes (Rhizoctoniasolani) for soil passes fungus diseases, flax anthrax-bacilus (Colletotrichumlinicola), point sickle spore flax specialized form (FusariumoxysporumSchltdl.exSnyderetHansenf.sp.lini), phytophthora blight of pepper (Phytophthoracapsici), tomato gray mould bacterium (Botrytiscinerea), Fusarium oxysporum (Fusariumoxysporum), Fusarium graminearum (F.graminearum), fusarium moniliforme (F.verticillioides), Rhizoctonia cerealis (R.cerealis), early epidemic germ (Alternariasolani), the soil of gaeumannomyce bacterium (Gaeumannomycesgraminis) or sclerotinite (Sclerotiniasclerotiorum) etc. passes fungus diseases.
The embodiment of the present invention has carried out bacillus amyloliquefaciens IBFCBF-1 and fermented liquid thereof to the research of fungal disease for phytophthora blight of pepper, and research contents is as follows.
(1) the selecting of substratum
The cultivation of bacillus amyloliquefaciens IBFCBF-1 adopts NB liquid nutrient medium (i.e. beef extract-peptone liquid nutrient medium), the preparation of this NB liquid nutrient medium comprises the peptone of 10g, the extractum carnis of 3g and the NaCl of 5g, above-mentioned substance uses deionized water to be settled to 1000ml, and adjust ph is 7.2-7.4, then 1 × 10
5the sterilized under pressure 20min of Pa, obtained NB liquid nutrient medium.
Capsicum epidemic disease enzymophathy fungal pathogens adopts PDA liquid nutrient medium, and the preparation of this PDA liquid nutrient medium comprises the peeled potatoes of 200g, the glucose of 20g, and above-mentioned substance uses deionized water to be settled to 1000mL, and adjust ph is 7.2-7.4, then 1 × 10
5the sterilized under pressure 20min of Pa, obtained PDA liquid nutrient medium.
(2) selection of capsicum variety
The Sweet Pepper Varieties eggplant door that capsicum selects Vegetable Research Inst., Hunan Prov. Agriculture Science Academy to provide.The hydrogen peroxide being 10% by the seed concentration of eggplant door carries out surface sterilization 20min, then uses sterilized water shower 3 times, and is positioned over
culture dish in, be constant temperature half-light vernalization under the condition of 30 DEG C in temperature.Choosing the long seed for 0.5cm of bud is sowed in 9 hole alms bowls, and matrix is commercially available Nutrition Soil, and the growth cabinet that capsicum is placed in 30 ± 1 DEG C is cultivated, and the intensity of now illumination is 12000Lux, and periodicity of illumination is L ︰ D=14 ︰ 10, RH=80% ± 10%.
(3) specific experiment design
Experiment is divided into 3 groups:
CK1: clear water; CK2:NB substratum; T: bacillus amyloliquefaciens IBFCBF-1 bacterium liquid.
Wherein, bacillus amyloliquefaciens IBFCBF-1 is 30 DEG C in temperature, and rotating speed is cultivate 48h under the condition of 180r/min, then with sterilized water, bacterium liquid is diluted to 10
5cFU/mL.Capsicum elicitin pathogenic bacteria is 30 DEG C in temperature, and rotating speed is cultivate 7d under the condition of 180r/min, then with sterilized water, bacterium liquid is diluted to 10
5cFU/mL.
When testing, the pepper seed two sprouted is sowed in 9 Bo Zhongmei hole, holes, ensure 18 strain young plant survivals, and 18 strain young plants are divided into three groups, when capsicum is cultured to the 3-4 leaf phase, capsicum elicitin pathogenic bacteria bacterium liquid 2.5mL is applied, then to being divided into the clear water, NB substratum and the bacillus amyloliquefaciens IBFCBF-1 bacterium liquid that add 2.5mL in the young plant of three groups respectively at the rhizosphere of capsicum.Apply 5mL nutritive medium every the every hole of 2d, to ensure that matrix is comparatively moistening, capsicum can normal growth.Observe chili growth situation, after 40d, capsicum carried out that plant height, stem are thick, the mensuration of interval, upper part fresh weight and dry weight and the growth indexes such as root system fresh weight and dry weight, statistics often organizes capsicum epidemic disease morbidity strain number.
(4) measuring method
Be plant height from pepper plant base portion to the distance between stem top and trunk diameter growth point during measurement, the diameter of the first segment stem of the nearly root knot of plant is that stem is thick.The length of the every trifle stem of plant is interval, and the first-half of nearly root knot is chosen in this experiment.First node of pepper plant from nearly root knot is cut off, takes the weight of upper part and root system part respectively, be designated as fresh weight, be then placed in the baking oven of 85 DEG C and dry to constant weight, again take its weight respectively, be designated as their dry weight.Experimental result please refer to table 2, table 3 and accompanying drawing 8.
Table 2: three kinds of different treatment methods are on the impact of chili growth
Table 3: a situation arises with the capsicum epidemic disease of three kinds of different methods process
| Process | Morbidity strain number (strain) | Sickness rate (%) | Prevention effect (%) |
| 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/investigate total strain number × 100; Prevention effect %=(1-treatment group diseased plant rate/control group diseased plant rate) × 100.
Can find out from table 2, the process of T group makes capsicum have higher plant height, thicker stem, and first segment pitch, second section pitch, upper part fresh weight, upper part dry weight, root fresh weight and root dry weight data are all large than all the other data of two groups.Meanwhile, can find out that pepper plant grows fine from accompanying drawing 8, thus, the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides and fermented liquid thereof effectively can promote the growth of plant.Can find out from table 3, the process of T group makes the sickness rate of capsicum be starkly lower than the sickness rate of other two groups, and prevention effect is obviously greater than the prevention effect of other two groups especially, can illustrate that the bacillus amyloliquefaciens IBFCBF-1 that the embodiment of the present invention provides and fermented liquid thereof have significant antagonistic effect thus.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a bacillus amyloliquefaciens bacterial strain, it is characterized in that, described Bacillus amyloliquefaciens strain is bacillus amyloliquefaciens IBFCBF-1, and described bacillus amyloliquefaciens IBFCBF-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.11230.
2. Bacillus amyloliquefaciens strain according to claim 1, is characterized in that, the bacterium colony of described bacillus amyloliquefaciens IBFCBF-1 is creamy white, translucent, and circular, edge is irregular, and surface wettability is smooth.
3. the preparation method of bacillus amyloliquefaciens IBFCBF-1 according to claim 2, is characterized in that, described preparation method comprises the following steps:
S01: gather soil sample by multi-point sampling method, and the soil sample porphyrize that will collect;
S02: the soil sample of porphyrize is put into the centrifuge tube that sterilized water is housed and fully shakes, obtain Soil Slurry;
S03: by described Soil Slurry gradient dilution, obtain the Soil Slurry of different concns;
S04: choosing concentration is 10
-5, 10
-6with 10
-7soil Slurry, and join and NA culture medium flat plate carry out coating process, be put in the incubator of 30 DEG C and cultivate, obtain bacterium colony;
S05: select single bacterium colony that form differs and carry out line conservation, cultivate 24 hours, obtain isolate, the refrigerator that described isolate is placed in 4 DEG C is saved backup;
S06: center pathogenic bacteria bacterium block being inoculated into PDA culture medium flat plate, inoculating described isolate, 25 DEG C of cultivations apart from plate center equidistant, that selection inhibition zone is maximum is bacillus amyloliquefaciens IBFCBF-1.
4. want the application of the bacillus amyloliquefaciens IBFCBF-1 in 1-3 described in any one according to right, it is characterized in that, the fermented liquid of described bacillus amyloliquefaciens IBFCBF-1 and described bacillus amyloliquefaciens IBFCBF-1 is applied to control fungal disease.
5. the application of bacillus amyloliquefaciens IBFCBF-1 according to claim 4, is characterized in that, described fungal disease is that soil passes fungus diseases.
6. the application of bacillus amyloliquefaciens IBFCBF-1 according to claim 5, it is characterized in that, it is dry thread Pyrenomycetes, flax anthrax-bacilus, sharp sickle spore flax specialized form, phytophthora blight of pepper, tomato gray mould bacterium, Fusarium oxysporum, Fusarium graminearum, fusarium moniliforme, Rhizoctonia cerealis, early epidemic germ, gaeumannomyce bacterium or sclerotinite that described soil passes fungus diseases.
7. the application of bacillus amyloliquefaciens IBFCBF-1 according to claim 4, it is characterized in that, the preparation of the fermented liquid of described bacillus amyloliquefaciens IBFCBF-1 comprises: be seeded in liquid fermentation medium by bacillus amyloliquefaciens IBFCBF-1, fermentation culture is carried out at 25-30 DEG C, shaking table concussion 2-4 days, obtains the fermented liquid of bacillus amyloliquefaciens IBFCBF-1.
8. the application of bacillus amyloliquefaciens IBFCBF-1 according to claim 7, is characterized in that, the rotating speed of described shaking table concussion is 180r/min.
9. the application of bacillus amyloliquefaciens IBFCBF-1 according to claim 8, is characterized in that, described liquid fermentation medium comprises following component according to concentration:
The Tryptones of 10g/L, the yeast of 5g/L leaches cream, the sucrose of 20g/L, the NaCl of 5g/L.
10. the application of bacillus amyloliquefaciens IBFCBF-1 according to claim 9, is characterized in that, the pH of described liquid fermentation medium is 7.2-7.4, and the sterilizing 20 minutes when temperature is 121 DEG C.
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