CN108893424A - A kind of bacillus megaterium BM22 and its microcapsule formulation and application - Google Patents
A kind of bacillus megaterium BM22 and its microcapsule formulation and application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus megaterium (Bacillus megaterium) BM22 and its microcapsule formulation and applications, the bacterial strain was preserved in China General Microbiological culture presevation administrative center on December 15th, 2017, and deposit number is CGMCC No.15070.It is the microcapsule formulation of active constituent by seed liquor culture using bacillus megaterium BM22, is dried to obtain after cultivation and fermentation in carrier, auxiliary agent and protectant mixture.Bacillus megaterium BM22 and its microcapsule formulation provided by the invention have good preventive effect to Chinese prickly ash bleeding disease, and 800-1600 times of dilution of Chinese prickly ash preparation of not falling ill is primary in spring smearing limb, can prevent the generation of Chinese prickly ash bleeding disease；Morbidity Chinese prickly ash annual spring, autumn use 400-800 times of dilution of preparation to smear application at scab after striking off gummosis, can effectively treat Chinese prickly ash bleeding disease.
Description
Technical field
The invention belongs to microorganisms technical fields, are related to a kind of bacillus megaterium more particularly to a kind of huge bud
Spore bacillus BM22 bacterial strain and its microcapsule formulation and the application in Chinese prickly ash bleeding disease.
Background technique
Chinese prickly ash is Rutaceae, Zanthoxylum machaka or dungarunga, is the main economic of xanthoxylum in world wide
Cultivar, in China, cultivation and usage history are long, and variety source is various, and cultivates in extensive range.Chinese prickly ash bioactive ingredients
It is various, medicinal, the edible and ecological value with higher.Its fruit is rounded, mung bean size, and crust is a kind of common perfume
Material, and extractable aromatic oil, and can be used as medicine, seed is edible, production soap also can be processed, while Chinese prickly ash is also arid and semi-arid
The important water and soil conservation tree species in mountain area.In medical value, in digestive system, cardiovascular system, suppression microbial action, anticancer
The development and application in the fields such as researchs and chemical industry, pesticide such as effect are extensive, have middle benefit gas promoting the circulation of qi, by cold, analgesic, desinsection and other effects；
The diseases such as stomach abdomen crymodynia, vomiting, diarrhea, blood fluke, roundworm can be controlled, and as epidermis anesthetic.
Chinese prickly ash bleeding disease is caused by fungi, has very strong infectiousness, and annual spray forms warty after catching an illness for the year
Protrusion, scab in May next year expand, sick body cracking overflow resin, just be semi-transparent clear flexible glue, after become dark brown crystalloid；
Perennial branch is caught an illness, and generates water vesicular protuberance and has gummy outflow.As germ encroaches on, aggrieved position necrosis causes limb withered
Extremely.There are the report of cause of disease, generation and harm, chemical prevention etc., but environmentally friendly, people and animals about Chinese prickly ash bleeding disease at present
Safety, sustainable use biological control be not directed to, related Chinese prickly ash bleeding disease biological control and its preparation research are still blank.
Summary of the invention
The technical problem to be solved by the present invention is to, in the defects of prevention and treatment Chinese prickly ash bleeding disease, provide for the prior art
Bacillus megaterium BM22 bacterial strain, microcapsule formulation and preparation method thereof and the application in Chinese prickly ash bleeding disease.
The present invention is realized especially by following technical scheme：
Bacillus megaterium (Bacillus megaterium) BM22 bacterial strain of the invention was used on June 15th, 2017
Dilution plating procedure is located away from Sichuan Dayi and looks up to heaven nest medicine field health Eucommia ulmoides Oliv. leaves, which reaches Chinese prickly ash gummosis germ bacteriostasis rate
To 98% or more, Institute of Microorganism, Academia Sinica's Chinese microorganism strain preservation is preserved on December 15th, 2017
Administration committee's common micro-organisms center (CGMCC), address are located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number
For CGMCC No.15070.
The single colonie that bacillus megaterium BM22 of the present invention has cultivated 2d on NA plating medium is creamy white, round,
Rough surface, edge waviness shape is opaque, inviscid.
The 16S rDNA sequence of bacillus megaterium BM22 of the present invention is as shown in SEQ ID NO.1.
The preparation method of the microcapsule formulation of bacillus megaterium BM22 of the present invention, includes the following steps：
1) bacillus megaterium fermentation liquid：Nutrition meat culture solution is after high pressure sterilization, by nutrient meat under the control of logical oxygen
Matter culture solution is contained in triangular flask, is inoculated with bacillus megaterium inclined-plane seed in germ-free condition, huge gemma is made in shaken cultivation
Bacillus fermentation liquid；
2) microcapsule formulation：Carrier, auxiliary agent and protective agent are packed into wide-angle bottle, sterile under the control of logical oxygen in proportion
State is inoculated with bacillus megaterium fermentation liquid, and inoculum concentration is the 10% of total amount, is uniformly mixed, using cornstarch as microcapsule formulation
Wall material, dry in Airstreamsprayingdrier after mixture is mixed with cornstarch (inlet air temperature is 155 DEG C, sample introduction flow
For 550mL/h, atomisation pressure 0.20MPa), bacillus megaterium microcapsule formulation is obtained.
Further, the nutrition meat culture solution is：Beef extract 7g, peptone 10g, NaCl 5g, water 1000mL.
Further, the auxiliary agent includes carrier, dispersing agent, wetting agent and stabilizer, the carrier, dispersing agent,
Wetting agent, stabilizer and protective agent percentage composition by weight is specially：Dispersing agent 8%, wetting agent 5%, stabilizer 6%, protection
Agent 4%, carrier supplies 100%.
Further, the carrier is white carbon black, and the dispersing agent is calcium lignosulfonate, and the wetting agent is
Polyethylene glycol, the stabilizer are oxalic acid, and the protective agent is riboflavin.
The performance indicator of the microcapsule formulation obtained by above-mentioned preparation method is：Beige powder, spherical partial size is in 3-
10 μm or so, moisture content 3.22%, ph6.98,325 mesh fineness percent of pass is 99%.
The bacillus megaterium BM22 microcapsule formulation that above-mentioned preparation method is prepared is also in protection scope of the present invention
Within.
The content of living spores is not less than 1 × 10 in the bacillus megaterium BM22 microcapsule formulation9cfu/g。
Application of the bacillus megaterium BM22 of the present invention in prevention and treatment Chinese prickly ash bleeding disease, it is certainly of the present invention
Application of the bacteria suspension or secondary metabolite of bacillus megaterium BM22 in prevention and treatment Chinese prickly ash bleeding disease is also in guarantor of the invention
It protects in range.
The present invention is preventing and treating answering in Chinese prickly ash bleeding disease by the microcapsule formulation of active constituent of bacillus megaterium BM22
With Chinese prickly ash of not falling ill is primary with inhibitor dilutions smearing limb in spring, and morbidity Chinese prickly ash uses preparation after spring, autumn strike off gummosis
It is each primary that dilution smears scab.
Beneficial effects of the present invention are：
Bacillus megaterium BM22 and its microcapsule formulation storage time provided by the invention are long, and survival rate is high, to Chinese prickly ash
Bleeding disease has good preventive effect, good stress resistance, and resistant to high temperatures, low temperature, resistance to drying are suitable for merchandized handling, nuisanceless, green ring
It protects, sustainable to control disease, production cost is low.
Detailed description of the invention
Fig. 1 is bacillus megaterium BM22rDNA-ITS sequence pcr amplification product electrophoretogram of the present invention；
Fig. 2 is the phylogenetic tree of the bacterial strain BM22 based on 16S rDNA sequence construct.
Specific embodiment
Technical solution of the present invention is further described below with reference to embodiment, it is as described below, it is only to of the invention
Preferred embodiment not limits the present invention, any person skilled in the art possibly also with
The technology contents of the disclosure above are changed or are modified as the equivalent embodiment changed on an equal basis.It is all without departing from the present invention program
Hold, according to the technical essence of the invention any simple modification, equivalent change and modification made to the above embodiment, all falls within this
In the protection scope of invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The separation and identification of 1 bacillus megaterium BM22 of embodiment
The separation of 1.1 bacillus megaterium BM22
Healthy Eucommia ulmoides Oliv. leaves are taken in Sichuan Dayi nest medicine field Cortex Eucommiae black spot generating region of looking up to heaven.The sample sterile water adopted back
It rinses well, weighs 1.0g tissue, 1-2min is impregnated in 70% alcohol, then sterilize 3- with 1-3% liquor natrii hypochloritis
5min, sterile water washing is several after, absorption 100 μ L of last time cleaning solution smearing NA plate (beef extract 3g, peptone 10g,
Sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7.0 mix 121 DEG C of high pressure sterilization 30min after packing), 27 DEG C
Dark culturing for 24 hours, for the control of surface sterilization, checks whether surface sterilization is thorough.
The blade of surface sterilization is shredded, sterile mortar is put into, the quartz sand and 10mL sterile water of sterilizing is added, fills
Divide grinding, stands 30min, dilution 10-1, 10-2, 10-3, 10-4, 10-5Totally 5 gradients draw 100 μ L 10 respectively-3, 10-4, 10-5This 3 gradient lapping liquids are respectively coated NA plate (beef extract 3g, peptone 10g, sodium chloride 5g, agar powder 15-20g, distillation
Water 1000mL, pH 7.0 mixes 121 DEG C of high pressure sterilization 30min after packing), 3 repetitions of each processing, 27 DEG C of culture 48h choose
The apparent single colonie of colonial morphology difference is taken to enter primary dcreening operation.
According to the size of bacterium colony, color, whether protrusion, whether edge feature, surface are smooth, the difference of transparency etc.
Different, for picking single colonie in the flat lining out purifying of NA, the inclined-plane NA of transferring after purification is spare in 4 DEG C of preservations.By 5 points of face-off methods,
PDA plate center inoculation diameter 5mm Chinese prickly ash bleeding disease pathogen bacteria cake, the equidistant above-mentioned bacterial strain to be screened of inoculation of surrounding, 25 DEG C
Constant temperature incubation, 3 repetitions of each processing.The size of the inhibition zone generated according to each screening bacterium determines antagonism degree, excellent effect
Bacterial strain (table 1) be BM22, antibacterial circle diameter reaches 21.2mm.
Inhibiting effect of 1 endogenetic bacteria of table to Chinese prickly ash gummosis germ (Phytophthora citrophthora)
The identification of 1.2 bacterial strains
It is huge in conjunction with physiological and biochemical index (table 2) and molecular biology identification endophyte BM22 through morphological observation
Bacillus.
2 bacterial strain BM22 physiological and biochemical index of table
The single colonie milky of the BM22 bacterial strain of 2d has been cultivated on NA plating medium, round, rough surface, edge wave
Line shape, it is opaque, it is inviscid.
Molecular biology identification (16S rDNA)：By extracting, DNA of bacteria carries out PCR amplification and electrophoresis, recovery product are sent
It is sequenced to biotech company；To be sequenced column in GenBank database it has been reported that sequence carry out homology BLAST point
Analysis, and multiple sequence comparison is carried out with Clustalx (1.83) software, then sent out with the adjacent method building system in Mega4.0 software
Tree is educated, determines status of the BM22 bacterial strain in microflora auxology.It is 949bp's that molecular biology identification, which obtains length,
The 16S rDNA sequence of BM22 bacterial strain is submitted GenBank database and carries out BLAST analysis, chosen wherein by DNA fragmentation (Fig. 1)
The higher bacterial 16 S rDNA sequence of homology therewith, and multiple matching arrangement analysis is carried out with Clustalx software, use Mega
It analyzes software building phylogenetic tree (Fig. 2).As can be seen that BM22 bacterial strain and JX286698 are with 99% 16SrRNA gene core
Nucleotide sequence similitude and higher value support of bootstrapping gather for 1 branch, and apart from each other with other bacillus, show BM22
It is nearest with the affiliation of Bacillus megaterium.
Based on features above, its classification naming of the strain is bacillus megaterium (Bacillus megaterium)
BM22 is stored in Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation conservator on December 15th, 2017
Meeting common micro-organisms center, deposit number are CGMCC No.15070.
The production of 2 bacillus megaterium BM22 microcapsule formulation of embodiment
Microcapsule formulation is made through everfermentation, drying in the bacillus megaterium BM22 obtained according to embodiment 1, specific to wrap
Include following steps：
1) fermentation liquid：It is (logical that nutrition meat culture solution is contained in 300mL triangular flask ratio after high pressure sterilization, in 100mL liquid
Oxygen control) bottling, the inclined-plane bacillus megaterium BM22 seed, 2 inclined-plane seeds of every bottle of inoculation, temperature are inoculated in germ-free condition
28-30 DEG C, bacillus megaterium BM22 fermentation liquid is made in initial pH value 6.5,120r/min shaken cultivation 36h.Wherein nutrient meat
Matter culture solution is：Beef extract 7g, peptone 10g, NaCl 5g, water 1000mL.
2) microcapsule formulation：By calcium lignosulfonate 8%, polyethylene glycol 5%, oxalic acid 6% is packed under the control of logical oxygen
300mL wide-angle bottle is inoculated with bacillus megaterium BM22 fermentation liquid in germ-free condition, and inoculum concentration is the 10% of gross mass, and mixing is equal
It is even；Using cornstarch as microcapsule formulation wall material, said mixture is mixed with cornstarch, white carbon black supplies 100%, after
(inlet air temperature is 155 DEG C, and sample introduction flow is 550mL/h, atomisation pressure 0.20MPa) dry in Airstreamsprayingdrier, system
At bacillus megaterium BM22 microcapsule formulation, then save.
Microcapsule formulation index：1×109Cfu/g living spores, Beige powder, spherical partial size are aqueous at 3-10 μm or so
Rate is that 3.22%, ph6.98,325 mesh fineness percent of pass are 99%.
Preparation saves：Sack drying is stored at room temperature 1.5 years or low temperature (4 degree) does not influence control efficiency in 3 years.
3 bacillus megaterium BM22 microcapsule formulation of embodiment tests the prevention of Chinese prickly ash bleeding disease
1) BM22 microcapsule formulation dilution prepares：1g microcapsule formulation obtained above is used 50 respectively, 100,200,
400,800,1600,2400mL dilution, are made dilution.
2) it does not fall ill Chinese prickly ash forest land, is smeared limb 1 time in annual spring with inhibitor dilutions in 1), 1-2 gives birth to 50mL, 3-
5 years raw 80mL, 5 years above 100-150mL of life are control with clear water.
3) 30d after smearing carries out disease survey statistics according to disease scale standard in table 3, and calculates disease incidence, and the state of an illness refers to
Several and control efficiency, the results are shown in Table 4.
3 Chinese prickly ash bleeding disease disease scale standard of table
Disease incidence (%)=(diseased plant number/strain number summation) × 100；
Disease index=[∑ (sick grade strain number × represent numerical value)/strain number summation × morbidity most heavy duty typical value] × 100；
Control efficiency (%)=[(control disease index growth rate-processing disease index growth rate)/control disease index increases
Long rate] × 100.
Table 4 do not fall ill Chinese prickly ash forest land application after preventive effect
After smearing prevention 30d, BM22 microcapsule formulation dilution shows good protecting effect to Chinese prickly ash bleeding disease, prevents
It is in a slight decrease with the increase of extension rate to control effect.From table 4, it can be seen that BM22 microcapsule formulation is diluted to 50-400 times of liquid
Control efficiency is all very high, has reached 100%, illustrates to fully control colonizing, grow, breeding for germ, disease does not occur.It is diluted to
2400 times of preventive effects are still above 60%.Therefore, consider from economic cost, BM22 microcapsule formulation is applied in prevention Chinese prickly ash bleeding disease
In generation, smearing 800-1600 times of liquid of application in spring once can reach preventive effect.
Experiment on therapy of the 4 bacillus megaterium BM22 microcapsule formulation of embodiment to Chinese prickly ash bleeding disease
1) BM22 microcapsule formulation dilution prepares：1g microcapsule formulation obtained above is used 50 respectively, 100,200,
400,800,1600,2400mL dilution, are made dilution.
2) it falls ill Chinese prickly ash forest land, is smeared respectively limb 1 time in annual spring, autumn with inhibitor dilutions in 1), 1-2 gives birth to
50mL, 3-5 give birth to 80mL, 5 years above 100-150mL of life, are control with clear water.
3) 30d after smearing carries out disease survey statistics according to disease scale standard in table 3, and calculates hair according to the above method
Sick rate, disease index and control efficiency, the results are shown in Table 5.
Therapeutic effect after the morbidity of table 5 Chinese prickly ash forest land application
After smearing treatment 30d, BM22 microcapsule formulation dilution (50-800 times) shows Chinese prickly ash bleeding disease good
Therapeutic effect, control efficiency are reduced with the increase of extension rate.As can be seen from Table 5, BM22 microcapsule formulation is diluted to 5 times
The control efficiency of liquid is very high, has been more than 90%, illustrates that disease occurs application BM22 later and can be effectively controlled the further expansion of cause of disease
Cause of disease is even killed in exhibition sprawling.800 times of liquid preventive effects are more than 50%, but are diluted to 1600-2400 times of liquid, under control efficiency is significant
It drops and is lower than 30.1%, therefore, consider from economic cost, BM22 microcapsule formulation is applied in treatment Chinese prickly ash bleeding disease,
It is once that can reach therapeutic effect that spring, autumn, it is each, which to smear 400-800 times of liquid of application,.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Sequence table
<110>Sichuan Agricultural University
<120>A kind of bacillus megaterium BM22 and its microcapsule formulation and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 949
<212> DNA
<213>Bacillus megaterium (Bacillus megaterium)
<400> 1
caggctgcgg ggctatacat gcagtcgagc gaactgatta gaagcttgct tctatgacgt 60
tagcggcgga cgggtgagta acacgtgggc aacctgcctg taagactggg ataacttcgg 120
gaaaccgaag ctaataccgg ataggatctt ctccttcatg ggagatgatt gaaagatggt 180
ttcggctatc acttacagat gggcccgcgg tgcattagct agttggtgag gtaacggctc 240
accaaggcaa cgatgcatag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt tgttagggaa 420
gaacaagtac gagagtaact gctcgtacct tgacggtacc taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540
gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactgggga acttgagtgc agaagagaaa agcggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcggctttt tggtctgtaa 720
ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaaga 949
Claims (10)
1. a kind of bacillus megaterium BM22, which is characterized in that the bacterial strain has been preserved on December 15th, 2017 Chinese common
Microbiological Culture Collection administrative center, deposit number are CGMCC No.15070.
2. a kind of bacillus megaterium BM22 according to claim 1, which is characterized in that the bacillus megaterium
The 16S rDNA sequence of BM22 is as shown in SEQ ID NO.1.
3. a kind of preparation method of bacillus megaterium BM22 microcapsule formulation, which is characterized in that include the following steps：
1) bacillus megaterium fermentation liquid：Nutrition meat culture solution trains nutrition meat under the control of logical oxygen after high pressure sterilization
Nutrient solution is contained in triangular flask, is inoculated with bacillus megaterium inclined-plane seed in germ-free condition, bacillus megaterium is made in shaken cultivation
Fermentation liquid；
2) microcapsule formulation：Carrier, auxiliary agent, protective agent are packed into wide-angle bottle, connect in germ-free condition under the control of logical oxygen in proportion
Kind bacillus megaterium fermentation liquid, inoculum concentration are the 10% of total amount, are uniformly mixed, using cornstarch as microcapsule formulation wall material,
It is dry in Airstreamsprayingdrier after mixture is mixed with cornstarch, obtain bacillus megaterium microcapsule formulation.
4. preparation method according to claim 3, which is characterized in that the nutrition meat culture solution is：Beef extract 7g,
Peptone 10g, NaCl 5g, water 1000mL.
5. preparation method according to claim 3, which is characterized in that the auxiliary agent includes carrier, dispersing agent, wetting agent
And stabilizer, the carrier, dispersing agent, wetting agent, stabilizer and protective agent percentage composition by weight are specially：Dispersing agent
8%, wetting agent 5%, stabilizer 6%, protective agent 4%, carrier supplies 100%.
6. preparation method according to claim 5, which is characterized in that the carrier is white carbon black, the dispersing agent
For calcium lignosulfonate, the wetting agent is polyethylene glycol, and the stabilizer is oxalic acid, and the protective agent is core yellow
Element.
7. the bacillus megaterium BM22 microcapsule formulation that preparation method as claimed in claim 3 is prepared.
8. microcapsule formulation according to claim 7, which is characterized in that the bacillus megaterium BM22 microcapsules system
The content of living spores is not less than 1 × 10 in agent9cfu/g。
9. the application of bacillus megaterium BM22 or its bacteria suspension or secondary metabolite in prevention and treatment Chinese prickly ash bleeding disease.
10. application of the microcapsule formulation as claimed in claim 7 in prevention and treatment Chinese prickly ash bleeding disease.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN111909877A (en) * | 2020-08-21 | 2020-11-10 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of chestnut branch blight |
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| CN110723920A (en) * | 2019-11-08 | 2020-01-24 | 东南大学 | Wetting agent for bacillus powder and preparation method thereof |
| CN111849843A (en) * | 2020-08-21 | 2020-10-30 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of bamboo shoot dry blight |
| CN111909877A (en) * | 2020-08-21 | 2020-11-10 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of chestnut branch blight |
| CN111925963A (en) * | 2020-08-21 | 2020-11-13 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease |
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