CN106577766B - A kind of composition, the preparation method and the usage of biocontrol bacteria and pyraclostrobin - Google Patents
A kind of composition, the preparation method and the usage of biocontrol bacteria and pyraclostrobin Download PDFInfo
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- CN106577766B CN106577766B CN201611154190.8A CN201611154190A CN106577766B CN 106577766 B CN106577766 B CN 106577766B CN 201611154190 A CN201611154190 A CN 201611154190A CN 106577766 B CN106577766 B CN 106577766B
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- pyraclostrobin
- composition
- bacillus amyloliquefaciens
- tobacco
- powdery mildew
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
- A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
- A01N47/24—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof containing the groups, or; Thio analogues thereof
Abstract
The invention discloses the compositions of a kind of biocontrol bacteria and pyraclostrobin, preparation method and the usage, in which: pyraclostrobin-containing is 15 ~ 300 μ g/ml, and bacillus amyloliquefaciens X60 is 1 × 106~1×1010CFU/ml；95% pyraclostrobin raw medicine is made into 1 × 10 with methanol4The mother liquor of μ g/ml, 4 DEG C of cryo-conservations；Bacillus amyloliquefaciens X60 microbial inoculum finished product viable bacteria concentration is 1 × 1010‑1×1012CFU/ml；Pyraclostrobin is mixed with bacillus amyloliquefaciens X60 wet thallus, is configured to that pyraclostrobin-containing is 15-300 μ g/ml, bacillus amyloliquefaciens X60 is 1 × 106‑1×1010The composition of CFU/ml.The present invention has preferable control efficiency to tobacco Powdery Mildew in Tobacco, environmental-friendly.
Description
Technical field
The present invention relates to field of biotechnology, relate in particular to the composition of a kind of biocontrol bacteria and pyraclostrobin,
Also relate to the preparation method and the biocontrol bacteria and pyraclostrobin of the composition of the biocontrol bacteria and pyraclostrobin
Purposes of the composition in terms of preventing and treating Powdery Mildew in Tobacco.
Background technique
Powdery Mildew in Tobacco (white powder) is occurred on tobacco growing by Powdery Mildew in Tobacco bacterium Erysiphe
Fungal disease caused by cichoracearum DC.They all have intermittent and fulminant feature, and tobacco leaf is sent out when serious
Sick rate may be up to 90%, so that flue cured tobacco quality is declined 1~2 grade, the underproduction of grave illness area, the reduction of income are up to 50% or more.In recent years, tobacco
Powdery mildew is always to seriously threaten one of Major Diseases of tobacco leaf production.Therefore, extremely urgent to the prevention and treatment of Powdery Mildew in Tobacco.
For a long time, Powdery Mildew in Tobacco is prevented and treated mainly by chemical prevention medicament carbendazim, triazolone etc. in production.Due to
Germ has the characteristic that proliferative speed is high, hereditary variation is big and grade of fit is high, continuous for many years largely to make to fall ill using chemical agent
Opportunistic pathogen generates serious drug resistance to main prevention and treatment fungicide.It would therefore be highly desirable to find new disease control solution.Methoxyl group
In acrylate chemical bactericide pyraclostrobin be a kind of modern wide spectrum, efficiently, systemic fungicide, sterling is white to shallow
Cream-coloured tasteless crystalline solid, alias are that N-f2- [1- (4- chlorobenzene) -1H- pyrazole-3-yl oxygen methyl] benzene foretells N- methoxy carbamic acid
Methyl esters, 63.7-65.2 DEG C of fusing point, 20-25 DEG C of vapour pressure is 2.6 × 10-8Pa.Also it is not used for the prevention and treatment of tobacco diseases；Meanwhile it is micro-
Bacteria agent can compete nutrition, space site with pathogen, and induction plant generates disease resistance, and secondary metabolite is to pathogen
It is inhibited, to plant have growth-promoting functions, and green, environmental protection, safety, in the prevention and treatment of plant disease have it is important
Application value.
Summary of the invention
A kind of pair of tobacco Powdery Mildew in Tobacco provided it is an object of the invention to overcome disadvantages mentioned above has preferable anti-
Control effect, the composition of environmental-friendly biocontrol bacteria and pyraclostrobin.
Another object of the present invention is to provide the preparation methods of the biocontrol bacteria and the composition of pyraclostrobin.
A further object of the present invention is to provide the compositions of the biocontrol bacteria and pyraclostrobin in prevention and treatment tobacco white powder
The purposes of sick aspect.
The composition of a kind of biocontrol bacteria and pyraclostrobin of the invention, pyraclostrobin-containing are 15~300 μ g/ml,
Bacillus amyloliquefaciens X60 is 1 × 106~1 × 1010CFU/ml。
Wherein: bacillus amyloliquefaciens X60, morphological feature: generating single colonie after cultivating 24 hours on NA culture medium,
Bacterium colony is milky, round, and there is serration at edge, and median rise, surface is smooth, and thallus is short straight rod shape, and gram-positive bacteria can
Gemma is generated, it can extracellular proteinase, cellulase etc..
The strain was deposited in China typical culture collection center (address: Wuhan City, Hubei Province on August 13rd, 2013
Wuchang Luo Jia Shan, Wuhan University), deposit number: CCTCC M 2013375, title are as follows: bacillus amyloliquefaciens X60 (Bacillus
Amyloliquefaciens X60),
(1) separating step of bacillus amyloliquefaciens X60 (Bacillus amyloliquefaciens):
10g tobacco rhizosphere soil is weighed in clean beaker using " isolation by dilution method ", is added on 90ml sterile water, magnetic force stirs
Mixing mixes well it, stands and draws 0.5ml supernatant after twenty minutes in 15 × 150mm clean tube, addition 4.5ml is sterile
Water mix well 10-1Concentration mother liquor, then 0.5ml supernatant is drawn from the test tube after dilution, it adds 4.5ml sterile water and fills
Divide to mix and is made 10-2Strength solution is successively diluted to 10-9The soil liquid of various concentration is stand-by.It is dense that above-mentioned difference is drawn respectively
100 μ l of the soil liquid is spent, is spread evenly across on the NA plate of culture bacterium, 28 DEG C of dark culturings, the picking list after bacterium grows
Bacterium colony purifying.
(2) culture medium:
Beef extract-peptone solid medium (NA): beef extract 3g, peptone 10g, agar powder 16g, water 1000ml, pH
6.8~7.2.
Beef extract-peptone fluid nutrient medium (NB): beef extract 3g, peptone 10g, water 1000ml, pH 6.8~7.2.
(3) conservation and passage of strain:
With the test tube of 18 × 180mm, NA culture medium 9ml is contained, beveling, inoculated bacteria, 28 DEG C are cultivated 2-3 days, set 4 DEG C
It saves, transferred species is primary within 3 months.
Bacterium after purification is crossed culture on NA culture medium, chooses single colonie after bacterium grows, with the tooth of sterilizing
Bacterium single colonie is chosen to the test tube (15 × 150mm) equipped with 5ml NB culture medium is inner and shakes training for 24 hours by label, draws 700 μ l bacterium solutions extremely
2ml bacterial strain saves in pipe, then adds 700 μ l, 30% sterile glycerol to saving in pipe, is placed in -20 DEG C of refrigerator-freezers and saves for a long time.
(4) morphological feature of strain:
The bacterium formed bacterium colony on NA plate, 30 DEG C, cultivate 2d, 5~15mm of colony diameter, bacterium colony subcircular, white,
Translucent, smooth to moisten, slightly swell, thallus is short straight rod shape, is aerobic bacteria, and Gram-reaction is positive.Through Bacteria Identification hand
Volume description, in conjunction with Biolog qualification result and 16S rDNA the sequencing results, is denoted as bacillus amyloliquefaciens for this bacterial strain
(Bacillus amyloliquefaciens X60)。
(5) cultural character of strain
20~50 DEG C of cultivation temperature, 30~37 DEG C of optimum temperature range；Initial pH4.0~9.0 are cultivated, most suitable 6.0~
7.0；Culture salinity 0.5~5%, best 0.5%；Carbon source, which can be cultivated, glucose, sucrose, fructose, galactolipin, lactose, wheat
Bud sugar, two pool of fiber, starch, wherein glucose is best；Nitrogen source, which can be cultivated, beef extract, peptone, KNO3、NH4NO3、(NH4)2SO4And NaNO2, wherein beef extract and peptone are best.
A kind of preparation method of the composition of of the invention biocontrol bacteria and pyraclostrobin, comprising the following steps:
(1) 95% pyraclostrobin raw medicine is made into 1 × 10 with methanol4The mother liquor of μ g/ml, 4 DEG C of cryo-conservations, for use；
(2) it is shaken in NB culture solution and is centrifuged the solution starch bud of collection in 10 minutes under training bacillus amyloliquefaciens X60,10000rpm
Spore bacillus X60 wet thallus；By bacillus amyloliquefaciens X60 in NB culture solution 30 DEG C 180rpm shaken cultivation 36-48 hours, so
10000rpm is centrifuged 10 minutes afterwards, takes X60 wet thallus and preservative fluid for thallus, is that 1:50 is made into microbial inoculum, microbial inoculum by mass volume ratio
Finished product viable bacteria concentration is 1 × 1010-1×1012CFU/ml；
(3) pyraclostrobin is mixed with bacillus amyloliquefaciens X60 wet thallus, being configured to pyraclostrobin-containing is 15-
300 μ g/ml, bacillus amyloliquefaciens X60 are 1 × 106-1×1010The composition of CFU/ml.
The preparation method of the composition of above-mentioned biocontrol bacteria and pyraclostrobin, wherein beef extract-peptone Liquid Culture
The preparation method of liquid (NB) are as follows: beef extract 3g, peptone 10g add deionized water to 1L, pH 6.8~7.2, sterilize through 120 DEG C
After twenty minutes.
The preparation method of the composition of above-mentioned biocontrol bacteria and pyraclostrobin, wherein preservative fluid for thallus is 0.01mol/
40 nonionic surface active agent of Tween of L phosphate buffer, 0.001mol/L.
A kind of purposes of the composition of of the invention biocontrol bacteria and pyraclostrobin in terms of preventing and treating Powdery Mildew in Tobacco.
Compared with prior art, the present invention having apparent beneficial effect, as can be known from the above technical solutions: the present invention is by pyrrole
Azoles kresoxim-methyl is combined with efficient biocontrol microorganisms, and bacillus amyloliquefaciens are formulated with wet bacterium and culture presevation liquid, and thallus is protected
Hiding liquid is 0.01mol/L phosphate buffer and 40 nonionic surface active agent of Tween of 0.001mol/L；Pyrazoles ether bacterium
Ester is first made into 1 × 10 with methanol4The mother liquor of μ g/ml is diluted with the sterile water containing 0.01% polysorbate40 when use.The composition can
With Direct spraying tobacco leaf.The present invention is specific to the disease-prevention composition of Powdery Mildew in Tobacco exploitation.Show through live test
Bacillus amyloliquefaciens strain X60 and pyraclostrobin composition have good inhibiting effect to cigarette Powdery Mildew in Tobacco, and have
There is the prevention effect of long period.Disease-controlling effect is individually made than pyraclostrobin independent role and bacillus amyloliquefaciens
The control efficiency of used time is good.It is environmentally friendly since action site of the composition to pathogen is more, it can effectively solve tobacco white powder
The resistance problems that germ generates traditional pesticide control are conducive to the safe sustainable development of tobacco leaf.
Therefore, the prevention and treatment for being used for Powdery Mildew in Tobacco has wide development space, while being also that agricultural safety can be held
The needs of supervention exhibition.
Specific embodiment
Embodiment 1
A kind of preparation method of the composition of biocontrol bacteria and pyraclostrobin, comprising the following steps:
(1) isolated from flue-cured tobacco (Nicotiana tabacum L.) rhizosphere soil.Bacillus amyloliquefaciens X60 exists
Single colonie is generated after cultivating 24 hours on NA culture medium, bacterium colony is milky, and round, there are serration, median rise, surface light in edge
Sliding, thallus is short straight rod shape, and gram-positive bacteria can produce gemma, can extracellular proteinase, cellulase etc..
By bacillus amyloliquefaciens X60 NB culture solution (NB cultivate liquid making method are as follows: beef extract 3g, peptone 10g,
Add deionized water to 1L, pH 6.8~7.2, sterilize through 120 DEG C) 30 DEG C 180rpm shaken cultivation 36-48 hours, then
10000rpm is centrifuged 10 minutes, take X60 wet thallus and preservative fluid for thallus (preservative fluid for thallus be 0.01mol/L phosphate buffer,
40 nonionic surface active agent of Tween of 0.001mol/L), it is that 1:50 is made into microbial inoculum by mass volume ratio, microbial inoculum finished product is living
Bacteria concentration is 1 × 1010-1×1012CFU/ml；
(2) 95% pyraclostrobin raw medicine is made into 1 × 10 with methanol4The mother liquor of μ g/ml, 4 DEG C of cryo-conservations, for use.
(3) prepare pyraclostrobin-containing in final test concentration be 300 μ g/ml, X60 containing bacillus amyloliquefaciens be 1 ×
106The biocontrol bacteria of CFU/ml and the composition of pyraclostrobin.
Embodiment 2
A kind of preparation method of the composition of biocontrol bacteria and pyraclostrobin, comprising the following steps:
(1) by bacillus amyloliquefaciens X60, in NB culture solution, (NB cultivates liquid making method are as follows: beef extract 3g, peptone
10g adds deionized water to 1L, pH 6.8~7.2, sterilizes through 120 DEG C) 30 DEG C 180rpm shaken cultivation 36-48 hours, then
10000rpm is centrifuged 10 minutes, take X60 wet thallus and preservative fluid for thallus (preservative fluid for thallus be 0.01mol/L phosphate buffer,
40 nonionic surface active agent of Tween of 0.001mol/L), it is that 1:50 is made into microbial inoculum by mass volume ratio, microbial inoculum finished product is living
Bacteria concentration is 1 × 1010-1×1012CFU/ml；
(2) 95% pyraclostrobin raw medicine is made into 1 × 10 with methanol4The mother liquor of μ g/ml, 4 DEG C of cryo-conservations, for use；
(3) prepare pyraclostrobin-containing in final test concentration be 20 μ g/ml, X60 containing bacillus amyloliquefaciens be 1 ×
109The biocontrol bacteria of CFU/ml and the composition of pyraclostrobin.
Embodiment 3
A kind of preparation method of the composition of biocontrol bacteria and pyraclostrobin, comprising the following steps:
(1) by bacillus amyloliquefaciens X60, in NB culture solution, (NB cultivates liquid making method are as follows: beef extract 3g, peptone
10g adds deionized water to 1L, pH 6.8~7.2, sterilizes through 120 DEG C) 30 DEG C 180rpm shaken cultivation 36-48 hours, then
10000rpm is centrifuged 10 minutes, take X60 wet thallus and preservative fluid for thallus (preservative fluid for thallus be 0.01mol/L phosphate buffer,
40 nonionic surface active agent of Tween of 0.001mol/L), it is that 1:50 is made into microbial inoculum by mass volume ratio, microbial inoculum finished product is living
Bacteria concentration is 1 × 1010-1×1012CFU/ml；
(2) 95% pyraclostrobin raw medicine is made into 1 × 10 with methanol4The mother liquor of μ g/m, 4 DEG C of cryo-conservations, for use；
(3) prepare pyraclostrobin-containing in final test concentration be 15 μ g/ml, X60 containing bacillus amyloliquefaciens be 1 ×
1010The biocontrol bacteria of CFU/ml and the composition of pyraclostrobin.
Test example: bacillus amyloliquefaciens X60 made from embodiment 1-3 and pyraclostrobin composition are to Powdery Mildew in Tobacco
Inhibition test
The controlling experiment of powdery mildew is carried out in big Tanaka, to start Powdery Mildew in Tobacco scab sporadicly occur on prosperous growth phase tobacco leaf
When start to test.
Biocontrol microorganisms processing group I, by obtained 1 × 1010CFU/ml bacillus amyloliquefaciens microbial inoculum even spraying is to tobacco seedlings leaf
Piece surface, until being lost；
The pyraclostrobin of pyraclostrobin processing group II, 200 μ g/ml is spraying, until being lost；
Example 1 group closes object processing group (I), the pyraclostrobin and 1 × 10 of 300 μ g/ml6CFU/ml solves starch gemma bar
Bacteria composition is spraying, until being lost；2 compositions-treated group (II) of embodiment, the pyraclostrobin and 1 × 10 of 20 μ g/ml9CFU/
Ml bacillus amyloliquefaciens composition spray, until being lost；3 compositions-treated group (III) of embodiment, the pyrazoles ether bacterium of 15 μ g/ml
Ester and 1 × 1010CFU/ml bacillus amyloliquefaciens composition spray, until being lost；Carbendazim control treatment group IV, more than 80% bacterium
1000 times of clever wettable powder dilutions are spraying, until being lost；Blank control group V, spraying sterile water.Every processing handles tobacco seedlings 60
Strain.Tobacco leaf is fallen ill under natural conditions.Blade incidence is observed and recorded after 20 days, and investigates disease index, calculates preventive effect.
According to state of an illness statistical grade below, state of an illness statistics is carried out according to blade onset area:
0 grade: full leaf disease-free spot；
1 grade: lesion area accounts for 1% or less blade area；
3 grades: lesion area accounts for the 2%~5% of blade area；
5 grades: lesion area accounts for the 6%~10% of blade area；
7 grades: lesion area accounts for the 11%~20% of blade area；
9 grades: lesion area accounts for 21% of blade area or more.
Field trial shows that bacillus amyloliquefaciens X60 can be good at preventing with pyraclostrobin antimicrobial composition group
The generation of Powdery Mildew in Tobacco, control efficiency reach 93.13%
To the preventive effect of Powdery Mildew in Tobacco after the application of 2 each group of table 20 days
Claims (5)
1. a kind of purposes of the composition of biocontrol bacteria and pyraclostrobin in terms of preventing and treating Powdery Mildew in Tobacco, in which: the combination
Object pyraclostrobin-containing is 15 ~ 300 μ g/ml, and bacillus amyloliquefaciens X60 is 1 × 106~ 1×1010CFU/ml。
2. the composition of a kind of biocontrol bacteria as described in claim 1 and pyraclostrobin is in terms of preventing and treating Powdery Mildew in Tobacco
Purposes, in which: bacillus amyloliquefaciens X60, deposit number: CCTCC M 2013375.
3. the composition of a kind of biocontrol bacteria as claimed in claim 1 or 2 and pyraclostrobin is in prevention and treatment Powdery Mildew in Tobacco side
The purposes in face, in which: the preparation method of the composition, comprising the following steps:
(1) 95% pyraclostrobin raw medicine is made into 1 × 10 with methanol4The mother liquor of μ g/ml, 4 DEG C of cryo-conservations, for use；
(2) it is shaken in NB culture solution and is centrifuged the solution starch gemma of collection in 10 minutes under training bacillus amyloliquefaciens X60,10000 rpm
Bacillus X60 wet thallus；By bacillus amyloliquefaciens X60 in NB culture solution 30 DEG C 180rpm shaken cultivation 36-48 hours, then
10000rpm is centrifuged 10 minutes, takes X60 wet thallus and preservative fluid for thallus, is that 1:50 is made into microbial inoculum by mass volume ratio, microbial inoculum at
Product viable bacteria concentration is 1 × 1010-1×1012CFU/ml；
(3) pyraclostrobin is mixed with bacillus amyloliquefaciens X60 wet thallus, being configured to pyraclostrobin-containing is 15-300 μ
G/ml, bacillus amyloliquefaciens X60 are 1 × 106-1×1010The composition of CFU/ml.
4. use of the composition of biocontrol bacteria as claimed in claim 3 and pyraclostrobin in terms of preventing and treating Powdery Mildew in Tobacco
On the way, the wherein preparation method of beef extract-peptone liquid medium (NB) are as follows: beef extract 3g, peptone 10g add deionized water extremely
1L, pH 6.8~7.2, after twenty minutes through 120 DEG C of sterilizings.
5. use of the composition of biocontrol bacteria as claimed in claim 3 and pyraclostrobin in terms of preventing and treating Powdery Mildew in Tobacco
On the way, wherein preservative fluid for thallus is 0.01 mol/L phosphate buffer, 40 non-ionic surface of Tween of 0.001mol/L is lived
Property agent.
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| CN103918719A (en) * | 2013-01-15 | 2014-07-16 | 陕西汤普森生物科技有限公司 | High-efficiency sterilization composition containing brevibacillus laterosporus |
| CN104642389A (en) * | 2015-01-28 | 2015-05-27 | 中国水稻研究所 | Biocontrol bacterium and pyraclostrobin composition |
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