CN105733991B - A kind of Bacillus amyloliquefaciens strain and its application in prevention and treatment Powdery Mildew in Tobacco - Google Patents
A kind of Bacillus amyloliquefaciens strain and its application in prevention and treatment Powdery Mildew in Tobacco Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
Present invention separation screening from medicinal plant mappia foetida obtains the bacterial strain with antagonism, it is identified through microbial taxonomy, the Strain Designation is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) D1 bacterial strain, and obtaining one kind using the fermentation liquid of the bacterial strain can be to the microbial bacterial agent that Powdery Mildew in Tobacco is prevented and treated.The most significant feature of the present invention is: providing a kind of new bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain D1, the microbial bacterial agent of its fermentation liquid preparation has good preventive and therapeutic effect to Powdery Mildew in Tobacco, is microbial resources important in planting industry.
Description
Technical field
The present invention relates to microorganisms technical field, micro- life of a kind of Bacillus amyloliquefaciens strain and its preparation is particularly related to
Application of the object microbial inoculum in prevention and treatment Powdery Mildew in Tobacco.
Background technique
Tobacco (Nicotiana tabacum) belongs to Solanaceae, annual herb plant.Currently, national tobacco planting area
Up to 1,000,000 hm2- 120 ten thousand hm2, 1,600,000-220 ten thousand tons of yield of tobacco, rank the first in the world.Powdery Mildew in Tobacco (Erysiphe
Cichoracearum) it is a kind of Common Diseases, is commonly called as " upper ash ", " frost occurs ", by developing upwards under plant, diseased plant rate Chang Da
100%, fall ill the general 10%-20% of leaf rate, serious up to 60% or more.Its cardinal symptom shows themselves in that susceptible just generation is white
Toner shape dot, it is hickie that point, which expands, is covered with entire blade, and sick leaf becomes brown from green, rear withered, raw white powder layer, i.e. pathogen
Conidiophore, conidium.China's Powdery Mildew in Tobacco, which endangers serious province, mainly Yunnan, Guizhou etc., causes every year
Direct economic loss be more than 100,000,000 yuan.
Some chemical agents such as triazolone, hexaconazole etc. have the effect of certain prevention and treatment Powdery Mildew in Tobacco, but with change
Learn to farm medicine residual and environmental pollution the problems such as it is increasingly prominent, research and development and application are safe from harm to people, animals and plants and environment micro-
Bacteria agent receives significant attention.It is residual with less toxic or nontoxic, low-residual or zero using antagonistic microbe prevention and treatment corps diseases
Stay, low pollution the advantages that, be not likely to produce drug resistance, and have antagonism, antibacterial broad spectrum activity to pathogenic microorganism, carry out in this way
Screening, preparation method have already appeared, but also seldom to the research of the biological control of Powdery Mildew in Tobacco at present, especially to some
The research of medicinal plant need further to develop.The present invention is directed to filter out a kind of micro- life of efficient antagonism Powdery Mildew in Tobacco of energy
Object bacterial strain, and microbial bacterial agent is made, the harm of Powdery Mildew in Tobacco is reduced by the method that the anti-gemma of biology is controlled.
The research of bacillus amyloliquefaciens is increasing at present, and metabolite is abundant, generates a variety of surface reactive materials,
As patent CN101935633A (applying date 2010.07.06) discloses a kind of solution starch gemma bar of biosurfactant production
Crude oil and reduce greasy filth harm that recycling is deposited in sludge are realized in application of the bacterium in oil sludge elution；In addition such as patent
Bacillus amyloliquefaciens RL263 is disclosed in CN104630086A (applying date 2014.11.20) has efficient prevention and treatment to rice blast
Effect, patent CN103937727A (applying date 2014.05.06) disclose a kind of solution starch bud of anti-tobacco mosaic viruses
Spore bacillus Z5, patent CN104711209A (applying date 2015.02.09) disclose a kind of for preventing and treating the Xie Dian of tobacco bacterial wilt
Afnyloliquefaciens B001, these patents show it mainly study be applied to inhibit fungi and bacterial activity etc., and
To tobacco black shank bacterium, tobacco brown spot pathogen, tobacco rhizoctonia solani have inhibition, the bacillus amyloliquefaciens of prevention and treatment have see specially
Benefit application is open.But not yet find the bacillus amyloliquefaciens for having inhibition, prevention and treatment to Powdery Mildew in Tobacco germ, what the present invention developed
A kind of microbial bacterial agent can reach effectively preventing effect to Powdery Mildew in Tobacco.
Summary of the invention
The purpose of the present invention is reducing chemical pesticide to prevent and treat environmental pollution caused by Powdery Mildew in Tobacco, people and animals' disease, mention
For one kind bacillus amyloliquefaciens that separation obtains from medicinal plant mappia foetida (Nothapodytes pittosporoides)
Bacterial strain, and using the microbial bacterial agent of bacterial strain preparation, for the prevention and treatment to Powdery Mildew in Tobacco.
The technical scheme is that
Bacillus amyloliquefaciens strain of the present invention, be 2014 by Guizhou Prov. Biochemical Engineering Center, Guizhou University from medicine
It is obtained with separation in plant mappia foetida (Nothapodytes pittosporoides), is named as bacillus amyloliquefaciens
(Bacillus amyloliquefaciens) D1 bacterial strain, is preserved in China typical culture collection center (CCTCC), address
Are as follows: the Chinese Wuhan Wuhan University, the deposit date is on September 21st, 2015, deposit number was CCTCC NO:M2015528.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) D1 bacterial strain of the invention has the following properties:
Colony morphology characteristic: gram-positive bacteria, white opaque colony, rough surface, edge on LB culture medium
Irregularly, the not chromogenic element on LB culture medium, growth temperature are 28~30 DEG C.See attached drawing 1-1,1-2.
Physiological and biochemical property: catalase reaction is positive；Hydrolyze starch；Acetyl methyl carbinol is positive；Nitrate reduction
It is positive；D-Glucose is positive；Mannitol is positive.
Molecular biological characteristics: PCR is carried out to bacterium 16s rna gene sequence using bacterial universal primers 27F and 1492R
Pcr amplification product is completed to be sequenced, and sequence is submitted to obtain Genbank in NCBI by amplification by Li Fei Bioisystech Co., Ltd
Accession number is KU 761585.
The isolated culture method of bacillus amyloliquefaciens bacterium D1 of the present invention, which comprises the following steps:
(1) preparation of mappia foetida plant tissue: taking the root of fresh mappia foetida, rinses well under tap water；
(2) on superclean bench, material surface sterilization and Sterility testing: is washed 2 with the Tween-80 sterile water of 1-5 ‰
~3 times, then it is washed to non-foam with sterile, surface moisture is sucked with aseptic filter paper；2~4min, nothing are impregnated with 75% alcohol
Bacterium water rinses 2~3 times；0.1-0.5% mercuric chloride 1~3min of surface sterilization, it is rear to use aseptic water washing 3~4 times, collect last time
Flushing water carry out microorganism detection, it is sterile can, aseptic filter paper blots excessive moisture；It is big that material is cut or is cut into 4~6mm of diameter
Small tissue dissection；
(3) endogenetic bacteria isolates and purifies: the tissue dissection after step (2) disinfection is inoculated on PDA culture medium plate,
In 22-30 DEG C constant temperature incubation 1~3 day, selection has the bacterial clump of antagonism, its scribing line purifying 2~3 of picking to endogenetic fungus
It is secondary, then with the bacterium colony streak inoculation after purification of transfer needle picking in cultivate 2 in PDA solid slope culture medium, at 22-30 DEG C~
4d is observed and is saved in the refrigerator for be put in 0~5 DEG C without after pollution, posting label to get D1 bacterial strain.
More specifically, the isolated culture method of Bacillus amyloliquefaciens strain D1 of the present invention is:
(1) preparation of mappia foetida plant tissue: taking the root of fresh mappia foetida, rinses well under tap water；
(2) on superclean bench, material surface sterilization and Sterility testing: is washed 2~3 with 1 ‰ Tween-80 sterile water
It is secondary, then it is washed to non-foam with sterile, surface moisture is sucked with aseptic filter paper；2~4min, sterile water are impregnated with 75% alcohol
It rinses 2~3 times；0.1% mercuric chloride 1~3min of surface sterilization, it is rear to use aseptic water washing 3~4 times, collect last time flushing water into
Row microorganism detection, it is sterile can, aseptic filter paper blots excessive moisture；Material is cut or is cut into the tissue of diameter 4~6mm size
Dissection；
(3) endogenetic bacteria isolates and purifies: the tissue dissection after step (2) disinfection is inoculated on PDA culture medium plate,
In 28 DEG C constant temperature incubation 1~3 day, selection has the bacterial clump of antagonism to endogenetic fungus, picking its scribing line purifying 2~3 times,
Then 2~4d is cultivated in PDA solid slope culture medium with the bacterium colony streak inoculation after purification of transfer needle picking at 28 DEG C, is seen
It examines after not polluting, posts label and be put in 4 DEG C of refrigerator and save to get D1 bacterial strain.
The present invention also provides by bacillus amyloliquefaciens (Bacillus amyloliquefaciens) D1 bacterial strain and PD
A kind of microbial bacterial agent that culture medium culture obtains.
Specifically, a kind of microbial bacterial agent preparation method, comprising the following steps:
Step 1: by the bacillus amyloliquefaciens D1 strain inoculated of activation in the PD culture medium of 200mL, 20-32 DEG C,
100-200r/min shaking table culture 18-30h, obtains primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 26-36 DEG C, 100~150r/
Min fermented and cultured 24-60h, obtains secondary seed solution；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 26-36 DEG C, 100-200r/
24-60h is to get microbial bacterial agent for min fermented and cultured.
Further, microbial inoculum of the invention is obtained through the following steps:
Step 1: by the bacillus amyloliquefaciens D1 strain inoculated of activation in the PD culture medium of 200mL, 28 DEG C, 120r/
Min shaking table culture for 24 hours, obtains primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 32 DEG C, 120r/min fermentation training
48h is supported, secondary seed solution is obtained；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 32 DEG C, 150r/min fermentation training
48h is supported to get microbial bacterial agent.
Another object of the present invention is to provide Bacillus amyloliquefaciens strain D1 and its microbial bacterial agent in prevention and treatment tobacco
Application in powdery mildew.
Beneficial effects of the present invention:
(1) present invention is separated from the root of medicinal plant mappia foetida to a kind of Bacillus amyloliquefaciens strain D1,
Compared with for trying other bacterial strains, have the function of significantly inhibiting to Powdery Mildew in Tobacco, provides one kind for prevention and treatment Powdery Mildew in Tobacco
New microbial strains.
(2) the present invention provides the microbial bacterial agent of Bacillus amyloliquefaciens strain D1 and its fermentation process；Inoculating strain
Fermentation liquid have significant difference, see attached drawing 2-1,2-1；Manufactured microbial bacterial agent can be applied to vegetative bacteria and fungal disease
Prevention and treatment, the field experiment of " cloud and mist 87 " shows that microbial inoculum made of bacterial strain D1 significantly inhibits Powdery Mildew in Tobacco,
Antimicrobial spectrum is wide, and bacteriostatic activity is strong, adds new approach for the exploitation of microbial bactericide.
(3) have certain growth-promoting functions: compared with for trying other bacterial strains, tobacco white powder can effectively be inhibited by spraying the microbial inoculum
The generation of disease, plant height, fresh weight, the dry weight of tobacco seedlings have significant advantage, to improve the yield of tobacco.
(4) zero malicious no pollutions: microbial bacterial agent of the invention is made of bacillus amyloliquefaciens, PD culture medium, and preparation is held
Easily, nontoxic, harmless, control efficiency is good, conducive to the plantation of the organic Chinese medicine of green.
Detailed description of the invention
Fig. 1-1 is D1 bacterial strain Gram's staining characteristic pattern (10 × 100 times of amplification)；
Fig. 1-2 is D1 bacterial strain colony morphology characteristic figure (diameter 9cm culture dish)；
Fig. 2-1 is not to be inoculated with D1 bacterial strain fermentation liquor figure；
Fig. 2-2 is inoculation D1 bacterial strain PD fermentation liquid figure；
Fig. 3 is the tobacco figure of 5 after treatments of microbial bacterial agent for applying number D1-1；
Fig. 4 is the control treatment tobacco figure for applying clear water.
Specific embodiment
In order to further illustrate technical solution of the present invention, the present invention also been mades following part test, it is intended into one
It walks and illustrates the present invention, rather than limitation of the invention.
Test example: the Field information of bacillus amyloliquefaciens D1 microbial inoculum
Agro-biological engineering research institute, Guizhou University, Guizhou Province test greenhouse, by microbial bacterial agent of the present invention and other
Microbial bacterial agent carries out field trial to " cloud and mist 87 ", and the microbial bacterial agent is sprayed on tobacco leaf, is sky with clear water
White control, chemicals spray as a control group, tree-plot, 9 repetitions, the medicine of spray in every 7 days, spray 5 times altogether, count tobacco
The incidence of powdery mildew.
Test group 1-3: the microbial bacterial agent of application embodiment 4-6 preparation, number D1-1, D1-2, D1-3；
Test group 3-4: the microbial inoculum that bacterial strain H1, H2 that application is obtained from mappia foetida are prepared into, number H1, H2；
Control group 1: application 2% antimycoin aqua, 200 times of liquid；
Control group 2: application can spray 15% triadimefon, 1000 times of liquid；
Blank control group: the clear water of isodose is applied.Number is CK；
Investigation method: tree-plot records each processing tobacco leaf sum, the sick number of sheets at different levels, and is classified and is calculated according to sick leaf
Disease index and relative control effect.
Powdery mildew blade grade scale:
0 grade: disease-free spot；1 grade: lesion area accounts for entire 5% or less leaf area；3 grades: lesion area accounts for entire leaf area
6%~10%；5 grades: lesion area accounts for entire leaf area 11%~20%；7 grades: lesion area account for entire leaf area 21%~
40%；9 grades: lesion area accounts for entire 40% or more leaf area.
Disease index=[Σ (diseased plants at different levels or the number of sheets × disease grade value)/(investigating total strain or the number of sheets × highest disease grade value)]
×100
Relative control effect (%)=[(control disease index-processing disease index)/control disease index] × 100
Table 1 sprays different microorganisms microbial inoculum to " cloud and mist 87 " powdery mildew disease index (%)
Table 2 sprays different microorganisms microbial inoculum to " cloud and mist 87 " powdery mildew relative control effect (%)
Note: "-" is acted on without preventive effect.
Test result shows that the microbial bacterial agent that the present invention obtains has significant prevention and treatment effect to " cloud and mist 87 " powdery mildew
Fruit, control efficiency are shown in attached drawing 3,4.
The cultural method of embodiment 1:D1 bacterial strain
(1) preparation of mappia foetida plant tissue: taking the root of fresh mappia foetida, rinses well under tap water；
(2) surface sterilization and Sterility testing: on superclean bench, being washed material 2 times with 1 ‰ Tween-80 sterile water,
It is washed to non-foam with sterile again, sucks surface moisture with aseptic filter paper；4min, aseptic water washing 3 are impregnated with 75% alcohol
It is secondary；0.1% mercuric chloride surface sterilization 1min, it is rear to use aseptic water washing 3 times, it collects last time flushing water and carries out microorganism detection,
It is sterile can, aseptic filter paper blots excessive moisture；Material is cut or is cut into the tissue dissection of diameter 4mm size；
(3) endogenetic bacteria isolates and purifies: the tissue dissection after step (2) disinfection is inoculated on PDA culture medium plate,
In 22 DEG C constant temperature incubation 1 day, selection has the bacterial clump of antagonism to endogenetic fungus, picking its scribing line purifying 2 times, then with
The bacterium colony streak inoculation of transfer needle picking after purification is observed and is not polluted in PDA solid slope culture medium, cultivating 2d at 22 DEG C
Afterwards, it posts label and is put in 4 DEG C of refrigerator and save to get D1 bacterial strain.
The cultural method of embodiment 2:D1 bacterial strain
(1) preparation of mappia foetida plant tissue: taking the root of fresh mappia foetida, rinses well under tap water；
(2) surface sterilization and Sterility testing: on superclean bench, being washed material 3 times with 3 ‰ Tween-80 sterile water,
It is washed to non-foam with sterile again, sucks surface moisture with aseptic filter paper；2min, aseptic water washing 2 are impregnated with 75% alcohol
It is secondary；0.3% mercuric chloride surface sterilization 3min, it is rear to use aseptic water washing 4 times, it collects last time flushing water and carries out microorganism detection,
It is sterile can, aseptic filter paper blots excessive moisture；Material is cut or is cut into the tissue dissection of diameter 6mm size；
(3) endogenetic bacteria isolates and purifies: the tissue dissection after step (2) disinfection is inoculated on PDA culture medium plate,
In 28 DEG C constant temperature incubation 1 day, selection has the bacterial clump of antagonism to endogenetic fungus, picking its scribing line purifying 3 times, then with
The bacterium colony streak inoculation of transfer needle picking after purification is observed and is not polluted in PDA solid slope culture medium, cultivating 4d at 28 DEG C
Afterwards, it posts label and is put in 4 DEG C of refrigerator and save to get D1 bacterial strain.
The cultural method of embodiment 3:D1 bacterial strain
(1) preparation of mappia foetida plant tissue: taking the root of fresh mappia foetida, rinses well under tap water；
(2) surface sterilization and Sterility testing: on superclean bench, being washed material 3 times with 5 ‰ Tween-80 sterile water,
It is washed to non-foam with sterile again, sucks surface moisture with aseptic filter paper；3min, aseptic water washing 3 are impregnated with 75% alcohol
It is secondary；0.5% mercuric chloride surface sterilization 3min, it is rear to use aseptic water washing 4 times, it collects last time flushing water and carries out microorganism detection,
It is sterile can, aseptic filter paper blots excessive moisture；Material is cut or is cut into the tissue dissection of diameter 6mm size；
(3) endogenetic bacteria isolates and purifies: the tissue dissection after step (2) disinfection is inoculated on PDA culture medium plate,
In 30 DEG C constant temperature incubation 2 days, selection has the bacterial clump of antagonism to endogenetic fungus, picking its scribing line purifying 2~3 times, so
Afterwards 3d is cultivated, observation does not have in PDA solid slope culture medium with the bacterium colony streak inoculation after purification of transfer needle picking at 30 DEG C
After pollution, posts label and be put in 5 DEG C of refrigerator and save to get D1 bacterial strain.
Embodiment 4: the preparation of microbial bacterial agent
Step 1: the bacillus amyloliquefaciens D1 strain inoculated that activation is obtained according to embodiment 1-3 either method in
In the PD culture medium of 200mL, 20 DEG C, 100r/min shaking table culture 30h obtain primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 26 DEG C, 100r/min fermentation training
60h is supported, secondary seed solution is obtained；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 30 DEG C, 100r/min fermentation training
60h is supported to get microbial bacterial agent.
Embodiment 5: the preparation of microbial bacterial agent
Step 1: the bacillus amyloliquefaciens D1 strain inoculated that activation is obtained according to embodiment 1-3 either method in
In the PD culture medium of 200mL, 28 DEG C, 120r/min shaking table culture for 24 hours, obtain primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 32 DEG C, 120r/min fermentation training
48h is supported, secondary seed solution is obtained；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 32 DEG C, 150r/min fermentation training
48h is supported to get microbial bacterial agent.
Embodiment 6: the preparation of microbial bacterial agent
Step 1: the bacillus amyloliquefaciens D1 strain inoculated that activation is obtained according to embodiment 1-3 either method in
In the PD culture medium of 200mL, 32 DEG C, 200r/min shaking table culture 18h obtain primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 36 DEG C, 150r/min fermentation training
It supports for 24 hours, obtains secondary seed solution；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 36 DEG C, 200r/min fermentation training
It supports for 24 hours to get microbial bacterial agent.
Claims (3)
1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain D1 is in terms of preventing and treating Powdery Mildew in Tobacco
Using the bacterial strain D1 is preserved in China typical culture collection center (CCTCC), address are as follows: the Chinese Wuhan Wuhan University,
The deposit date is on September 21st, 2015, deposit number was CCTCC NO:M2015528.
2. numbering microbial bacteria made of Bacillus amyloliquefaciens strain D1 and the PD culture medium for being CCTCC NO:M2015528
Application of the agent in terms of preventing and treating Powdery Mildew in Tobacco, which is characterized in that the microbial inoculum the preparation method is as follows:
Step 1: by the bacillus amyloliquefaciens D1 strain inoculated of activation in the PD culture medium of 200mL, 20-32 DEG C, 100-
200r/min shaking table culture 18-30h, obtains primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 26-36 DEG C, 100~150r/min hair
Ferment culture 24-60h, obtains secondary seed solution；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 26-36 DEG C, 100-200r/min hair
24-60h is to get microbial bacterial agent for ferment culture.
3. application according to claim 2, which is characterized in that the microbial inoculum is obtained through the following steps:
Step 1: by the bacillus amyloliquefaciens D1 strain inoculated of activation in the PD culture medium of 200mL, 28 DEG C, 120r/min shakes
Bed culture for 24 hours, obtains primary seed solution；
Step 2: step 1 is obtained into primary seed solution, is inoculated in the PD culture medium of 10L, 32 DEG C, 120r/min fermented and cultured
48h obtains secondary seed solution；
Step 3: step 2 is obtained into secondary seed solution, is inoculated in the PD culture medium of 80L, 32 DEG C, 150r/min fermented and cultured
48h is to get microbial bacterial agent.
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| CN111218414A (en) * | 2019-11-16 | 2020-06-02 | 湖南省烟草公司湘西自治州公司 | Bacillus amyloliquefaciens, application and preparation method of fermentation liquor of bacillus amyloliquefaciens |
| CN111066821A (en) * | 2019-12-10 | 2020-04-28 | 中国农业科学院烟草研究所 | Application of bacillus amyloliquefaciens in prevention and treatment of tobacco powdery mildew and tobacco black shank and microbial inoculum thereof |
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