CN103114055B - Bacillus subtilis 9A and application thereof - Google Patents
Bacillus subtilis 9A and application thereof Download PDFInfo
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- CN103114055B CN103114055B CN201310020944.0A CN201310020944A CN103114055B CN 103114055 B CN103114055 B CN 103114055B CN 201310020944 A CN201310020944 A CN 201310020944A CN 103114055 B CN103114055 B CN 103114055B
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Abstract
The invention relates to bacillus subtilis 9A and application thereof. The bacillus subtilis 9A has the collection number of CCTCC No. M2012493, and a method for preparing a bacillus subtilis 9A agent is provided. The bacillus subtilis 9A and the agent thereof have the effects of inhibiting pathogenic bacteria growth of mango anthracnose, has the powerful inhibition effect even killing effect on the conidiospore, a protective film layer can be formed on the surface of an application object to prevent the pathogenic bacteria from intruding, the occurrence rate of mango anthracnose is effectively reduced, and the bacillus subtilis 9A does not have harm to mango, is environment-friendly and has good biopesticide development prospect.
Description
Technical field
The present invention relates to agriculture microbial technology field, relate to particularly a kind of subtilis 9A and application thereof.
Background technology
Colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides) causes one of Major Diseases that Mango fruit anthrax Shi Mango fruit produces, and all there is generation in Ge Mango fruit producing region in the world.
Blade tip, flower fringe and the fruit of this germ energy Qin Ran Mango fruit, cause top drying, tikka, fallen leaves, fallen flowers and shedding etc., and this bacterium has latent infection characteristic, after adopting, the storing phase can cause that blackspot piece and fruit rot appear in pericarp, greatly shortens the shelf-lives of Shang Pin Mango fruit.
This disease mainly adopts chemical agent to prevent and treat at present, but life-time service chemical pesticide can make pathogenic bacteria develop immunity to drugs, and brings the safety problem of contaminate environment and pesticide residue, and therefore, the biocontrol microorganisms that development and utilization nature exists has important practice significance.Bacterium is widely distributed at the earth, of a great variety, and aboundresources utilizes antagonistic bacterium to have obvious resources advantage as control of crop disease material.
Summary of the invention
The object of this invention is to provide subtilis and microbial inoculum that a strain can be used for Fang Zhi Mango fruit anthrax.
Another object of the present invention is to provide the application in control plant anthrax of above-mentioned subtilis and microbial inoculum thereof.
Subtilis 9A(Bacillus subtilis of the present invention) on December 3rd, 2012 be preserved in Chinese Typical Representative culture collection center (be called for short CCTCC; Address: China. Wuhan. Wuhan University; Postcode: 430072), deposit number CCTCC No.M2012493.
The viable count of subtilis 9A microbial inoculum provided by the invention is 10
9cfu/ml.Preferably 3-8 × 10
9cfu/ml.
The preparation method of described subtilis 9A microbial inoculum, comprises the following steps:
(1) bacterial strain activation: subtilis 9A is inoculated on solid medium, cultivates 1-2 days at 25 ℃-28 ℃;
(2) ferment-seeded is cultivated: by above-mentioned activation inoculation, in liquid nutrient medium, shaking culture 12-24 hour at 25 ℃-28 ℃, makes seed liquor;
(3) bacteria fermentation: above-mentioned seed liquor is inoculated in bacteria fermentation nutrient solution, and shaking culture 48-72 hour at 25 ℃-28 ℃, makes the microbial inoculum of subtilis 9A.
Bacterial strain activation culture by solid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, agar powder 15g, pH7.0-7.2.
Strain fermentation seed culture by liquid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, pH7.0-7.2.
Bacteria fermentation nutrient solution formula: Sucus Cucurbitae moschatae (200g pumpkin liquor) 1000mL, glucose 10g, peptone 10g.
Wherein, in step (3), the inoculum size of seed liquor is 0.05-0.1%(volume percent).
Subtilis 9A provided by the invention and fermented liquid microbial inoculum Dui Mango fruit anthrax germ mycelia thereof have the conidium that suppresses growth and teratogenesis ， Dui Mango fruit anthrax germ to have inhibition to sprout and lethal effect.The microbial inoculum of subtilis 9A provided by the invention is for plant anthrax, and it is the control of Mango fruit anthrax especially.
The invention has the beneficial effects as follows:
Subtilis 9A of the present invention and microbial inoculum Dui Mango fruit anthrax pathogen growth thereof have restraining effect; its conidium is had to even lethal effect of strong restraining effect; on subject surface, can form layer protecting film stops germ to be invaded; the generation of You effect Jian Shao Mango fruit anthrax; and Dui Mango fruit is without poisoning; environmentally safe, has good biological pesticide DEVELOPMENT PROSPECT.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of subtilis 9A on PDA flat board;
Fig. 2 is transmission electron microscope observing subtilis 9A thalli morphology;
Fig. 3 is the restraining effect of subtilis 9A fermentating liquid filtrate Dui Mango fruit anthrax-bacilus mycelial growth; A Wei Mango fruit anthrax-bacilus mycelia normal growth situation, B is the growing state of anthrax-bacilus mycelia while being mixed with 9A culturing filtrate;
Fig. 4 is the impact of subtilis 9A fermentating liquid filtrate on Mango fruit anthrax-bacilus mycelia form; A is anthrax-bacilus normal bacteria filament shapes, and B is 9A culturing filtrate mycelia form after treatment;
Fig. 5 is the conidial germination inhibitor of subtilis 9A fermentating liquid filtrate Dui Mango fruit anthrax-bacilus; A is the normal anthrax-bacilus spore of sprouting; B is for using 9A culturing filtrate anthrax-bacilus spore after treatment;
Fig. 6 is the prevention effect of the in vitro Fruit Anthracnose of subtilis 9A fermented liquid Dui Mango fruit; A is the in vitro fruit of sterilized water processing; B is the in vitro fruit of 9A microbial inoculum processing;
Fig. 7 is the prevention effect of subtilis 9A fermented liquid Dui Mango fruit excised leaf anthrax; A is the excised leaf that sterilized water is processed contrast; B is the excised leaf of 9A microbial inoculum processing.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Separation, screening and the evaluation of embodiment 1 subtilis 9A
The separation of 1.1 bacteriums
With the writing brush through sterilizing, on melon leaf Powdery Mildew scab, gently sweep, sweep junk and fall within on PDA culture medium flat plate, be placed in 26 ℃ of natral light cahures of incubator.Routine observation between incubation period, carries out purifying to the bacterial colony of mode of appearance color notable difference, saves backup.
PDA culture medium prescription: potato 200g, glucose 10g, agar 16g, distilled water 1000ml.
The screening of 1.2 antagonistic strain 9A obtains
Primary dcreening operation: adopt dull and stereotyped face-off growth method using Yi Zhu Mango fruit anthrax-bacilus as indicator on PDA flat board to 1.1 in each bacterial isolates of obtaining of separation and purification carry out antagonism screening, the bacterial strain with antagonistic action is saved backup.
Multiple sieve: the antagonistic strain that primary dcreening operation is obtained is cultivated 48-72h on beef extract-peptone solid medium, getting each lawn one encircles to access respectively and in 100mL seed culture fluid, cultivates 12-48h and make seed liquor, draw respectively again 100 μ L seed liquor and inject 150ml bacteria fermentation nutrient solution, be placed in 28 ℃ of shaking tables, 180r/min condition cultivation 48-120h.Get each nutrient solution in the centrifugal 10min of 10000r/min, get supernatant liquor and obtain supernatant fluid filtrate through 0.22 μ m membrane filtration, draw each supernatant fluid filtrate and temperature and be about 45 ℃ of PDA substratum flat board that to be mixed containing supernatant fluid filtrate concentration be 8%, adopt growth rate method to filter out the strongest bacterial strain of Dui Mango fruit anthrax-bacilus mycelial growth restraining effect, obtain aimed strain 9A.
Beef extract-peptone solid culture based formulas: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, agar powder 15g, pH7.0-7.2.
Seed culture liquid formula: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, pH7.0-7.2.
Bacteria fermentation nutrient solution formula: Sucus Cucurbitae moschatae (200g pumpkin liquor) 1000mL, glucose 10g, peptone 10g.
The evaluation of 1.3 subtilis 9A
The result of morphologic observation shows, bacterial strain 9A is bacteria colony white on PDA substratum, ellipse, and neat in edge, surface drying is opaque.Transmission electron microscope observing result shows, 9A thalline is shaft-like, peritrichous.Physiology and biochemistry measurement result is the catalase test positive, v-p tests positive, Starch Hydrolysis enzyme positive, methyl red test feminine gender, phenylalaninase negative, tyrosine oxidase negative, D-Glucose L-arabinose D-wood sugar PEARLITOL 25C produces sour positive aerogenesis feminine gender, 10%NaCL grows positive, 50 ℃ of growths are negative, 45 ℃ of growths are positive, 10 ℃ of growths are positive, 5 ℃ of growths are negative, motility test is positive, cellulase feminine gender, the nitrate reduction positive, anti-nitrate reductase feminine gender, oxidation of ethanol feminine gender, lipase (tween 80) feminine gender, egg fat enzyme positive.16SrDNA the sequencing results shows, the 16SrDNA sequence of this bacterium and the subtilis that some have been reported have higher homology.Based on the above results, bacterial strain 9A is accredited as to subtilis (Bacillus subtilis).
The preparation of embodiment 2 subtilis 9A fermented liquids
(1) bacterial strain activation: subtilis 9A is inoculated on solid medium, cultivates 1 day at 25 ℃-28 ℃;
(2) ferment-seeded is cultivated: the lawn of above-mentioned activation bacterial strain one ring is inoculated in the seed culture liquid substratum of 100ml, and at 25 ℃-28 ℃, 180r/m shaking culture 12 hours, makes seed liquor;
(3) bacteria fermentation: press 0.05%(V/V) inoculum size, above-mentioned seed liquor is inoculated in bacteria fermentation nutrient solution, 180r/m shaking culture 48 hours at 25 ℃-28 ℃, (viable count is about 3.0 × 10 to make the microbial inoculum of subtilis 9A
9cfu/ml).
Bacterial strain activation culture by solid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, agar powder 15g, pH7.0-7.2.
Strain fermentation seed culture by liquid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, pH7.0-7.2.
Bacteria fermentation nutrient solution formula: Sucus Cucurbitae moschatae (200g pumpkin liquor) 1000mL, glucose 10g, peptone 10g.
The preparation of embodiment 3 subtilis 9A fermented liquids
(1) bacterial strain activation: subtilis 9A is inoculated on solid medium, cultivates 2 days at 25 ℃-28 ℃;
(2) ferment-seeded is cultivated: the lawn of above-mentioned activation bacterial strain one ring is inoculated in the seed culture liquid substratum of 100ml, and at 25 ℃-28 ℃, 180r/m shaking culture 24 hours, makes seed liquor;
(3) bacteria fermentation: press 0.1%(V/V) inoculum size, above-mentioned seed liquor is inoculated in bacteria fermentation nutrient solution, 180r/m shaking culture 72 hours at 25 ℃-28 ℃, (viable count is about 8.0 × 10 to make the microbial inoculum of subtilis 9A
9cfu/ml).
Bacterial strain activation culture by solid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, agar powder 15g, pH7.0-7.2.
Strain fermentation seed culture by liquid culture based formulas is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, pH7.0-7.2.
Bacteria fermentation nutrient solution formula: Sucus Cucurbitae moschatae (200g pumpkin liquor) 1000mL, glucose 10g, peptone 10g.
The impact of embodiment 4 subtilis 9A fermentating liquid filtrates on Mango fruit anthrax-bacilus mycelial growth and spore germination
The restraining effect of 4.1 subtilis 9A fermentating liquid filtrate Dui Mango fruit anthrax-bacilus mycelial growths
On PDA flat board, punch, the fermentating liquid filtrate that the centrifuged supernatant of subtilis 9A fermented liquid prepared by injection embodiment 2 obtains through 0.22 μ m membrane filtration, in apart from the Jie Shang of 25mm place, hole Mango fruit anthrax-bacilus bacterium piece, in 26 ℃ of incubators, cultivate, when there is antibacterial band, picking antibacterial with on mycelia in micro-Microscopic observation.As shown in Figure 3, Figure 4, result shows, subtilis 9A fermentating liquid filtrate can suppress anthrax-bacilus mycelial growth, causes that anthrax-bacilus mycelia form changes as mycelia branch increases, distortion, expands etc.
The restraining effect of 4.2 subtilis 9A fermentating liquid filtrate Dui Mango fruit anthrax-bacilus conidia germinations
Getting fermentating liquid filtrate that the centrifuged supernatant of subtilis 9A fermented liquid prepared by embodiment 2 obtains through 0.22 μ m membrane filtration and temperature, to be about that 45 ℃ of PDA substratum are mixed containing fermentating liquid filtrate concentration be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 10% flat board, drawing conidial suspension 0.1mL is evenly coated with at planar surface, in 26 ℃ of incubators, cultivate, respectively at 8h, 18h, 36h, 72h, 240h, carry out observed and recorded, as shown in Figure 5, result shows that concentration is that 4% filtrate can be suppressed the conidial sprouting of anthrax-bacilus completely.
The prevention effect of embodiment 5 subtilis 9A fermented liquid Dui Mango fruit blade anthrax
5.1 subtilis 9A fermented liquids are measured the prevention effect of excised leaf anthrax
Getting the tender leaf of the close anosis health of size puts in basket, on blade face, middle vein both sides, with marking pen, respectively draw the circle of three diameter 3mm, on each circle, smear subtilis 9A fermented liquid prepared by embodiment 2, artificial inoculation Mango fruit anthrax-bacilus conidial suspension after 24 hours.Every leaf is totally six circles, repeats 12 blades.With sterilized water and 1000 times of liquid of prochloraz aqueous emulsion (effective constituent 450g/L), replace subtilis 9A fermented liquid to compare, bagging moisturizing is cultivated in 26 ℃ of incubators, observes and manages incidence everywhere.As shown in Figure 6, result shows, after inoculation, the 5th day sterilized water control group sickness rate is 100%, and subtilis 9A fermented liquid and prochloraz treatment group have no morbidity.
The field controling test of 5.2 subtilis 9A fermented liquid Dui Mango fruit blade anthrax
In tree crown circumference all directions, choose at random without the tender tip of scab, spray subtilis 9A fermented liquid prepared by embodiment 2 evenly moistening to blade face, after 24 hours, Pen mist Jie Zhong Mango fruit anthrax-bacilus conidial suspension is evenly moistening to blade face, after being disposed, use the plastic bag cover tip, repeat 9 tips, with sterilized water and 1000 times of liquid of prochloraz aqueous emulsion (effective constituent 450g/L), replace subtilis 9A fermented liquid to compare, in the 7th day, from every tip, take out at random 5 leaf investigation incidences.Result shows, in rear the 7th day sterilized water control group disease index of inoculation, reaches 88.33%, and prochloraz group disease index is 20%, and protection effect is 77.36%, subtilis 9A fermented liquid group disease index average out to 15.00%, and protection effect is 83.02%.The preventive effect of subtilis 9A fermented liquid is significantly higher than the prevention effect of 1000 times of liquid of contrast medicament prochloraz.
The protection effect of embodiment 6 subtilis 9A fermented liquid Dui Mango fruits anthrax
6.1 subtilis 9A fermented liquids are measured the prevention effect of in vitro Fruit Anthracnose
Getting the tender fruit of the close anosis health of size puts in basket, in upward, simultaneously use the mark stroke circle of the about 3mm in footpath always, on each circle, smear subtilis 9A fermented liquid prepared by embodiment 2, artificial inoculation Mango fruit anthrax-bacilus conidial suspension after 24 hours, repeat 20 fruits, with sterilized water and 1000 times of liquid of prochloraz aqueous emulsion (effective constituent 450g/L), replace subtilis 9A fermented liquid to compare, bagging moisturizing is cultivated in 26 ℃ of incubators, observes fruit incidence.As shown in Figure 7, result shows, after inoculation, the 5th day sterilized water control group sickness rate is 100%, and subtilis 9A fermented liquid and prochloraz treatment group have no morbidity, and 1000 times of liquid of subtilis 9A fermented liquid and prochloraz are suitable to the prevention effect of in vitro Fruit Anthracnose.
The field controling test of 6.2 subtilis 9A fermented liquid Dui Mango fruits anthrax
Under tree crown week all directions choose at random surface without scab Chinese olive clear water clean dry, full fruit is smeared subtilis 9A fermented liquid to fruit vertex (vertices) prepared by embodiment 2 and occurs drop, after 24 hours, Pen mist Jie Zhong Mango fruit anthrax-bacilus conidial suspension to fruit surface is covered with spore liquid and till fruit vertex (vertices) forms drop and drips, after being disposed with plastic bag cover fruit, spacing between plastics bag and fruit, repeat 15 fruits, with sterilized water and 1000 times of liquid of prochloraz aqueous emulsion (effective constituent 450g/L), replace subtilis 9A fermented liquid to compare, observe incidence.Result shows, the 7th day sterilized water control group disease index average out to 94.08% after inoculation, and prochloraz group disease index average out to 20%, protection effect is 78.82%, subtilis 9A fermented liquid group disease index average out to 1.48%, protection effect is 98.41%.The protection effect of subtilis 9A fermented liquid is significantly higher than the prevention effect of 1000 times of liquid of contrast medicament prochloraz.
In embodiment 4-6, subtilis 9A fermented liquid prepared by embodiment 2 replaces with subtilis 9A fermented liquid prepared by embodiment 3, has same effect.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. subtilis (Bacillus subtilis) 9A, its preserving number is CCTCCNo.M2012493.
2. contain the microbial inoculum of subtilis 9A described in claim 1.
3. microbial inoculum according to claim 2, is characterized in that, the viable count of described microbial inoculum is 10
9cfu/ml.
4. the preparation method of microbial inoculum described in claim 2 or 3, comprises the following steps:
(1) bacterial strain activation: subtilis 9A is inoculated on solid medium, cultivates 1-2 days at 25 ℃-28 ℃;
(2) ferment-seeded is cultivated: by above-mentioned activation inoculation, in liquid nutrient medium, shaking culture 12-24 hour at 25 ℃-28 ℃, makes seed liquor;
(3) bacteria fermentation: above-mentioned seed liquor is inoculated in bacteria fermentation nutrient solution, shaking culture 48-72 hour at 25 ℃-28 ℃, makes the microbial inoculum of subtilis 9A, and described bacteria fermentation nutrient solution formula is: Sucus Cucurbitae moschatae 1000mL, glucose 10g, peptone 10g.
5. preparation method according to claim 4, is characterized in that, described in step (1), solid medium is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, agar powder 15g, pH7.0-7.2.
6. preparation method according to claim 4, is characterized in that, described in step (2), the liquid nutrient medium of seed culture is: extractum carnis 5.0g, peptone 10.0g, NaCl5.0g, distilled water 1000mL, pH7.0-7.2.
7. preparation method according to claim 4, is characterized in that, in step (3), the inoculum size of seed liquor is 0.05-0.1%.
8. the application of subtilis 9A in control plant anthrax described in claim 1.
9. the application of microbial inoculum in control plant anthrax described in claim 2 or 3.
10. application according to claim 8 or claim 9, is characterized in that, described plant anthrax is Mango fruit anthrax.
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| CN104164394B (en) * | 2014-08-04 | 2016-08-24 | 北京林业大学 | The bacterial strain of one strain antagonistic phytopathogen and application thereof |
| CN106544294A (en) * | 2016-10-12 | 2017-03-29 | 武汉骏安生物科技有限公司 | A kind of bacillus subtilises H 9 and its application |
| CN112266884B (en) * | 2020-10-28 | 2023-05-30 | 广西壮族自治区农业科学院植物保护研究所 | Application of bacillus subtilis N-42-2 in preventing and controlling mango anthracnose |
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| CN102643760B (en) * | 2011-02-18 | 2014-04-16 | 中国热带农业科学院环境与植物保护研究所 | Antagonistic bacterium capable of generating siderophore for controlling plant diseases |
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