CN109370939A - One plant of Bei Laisi bacillus and its separation method and application - Google Patents
One plant of Bei Laisi bacillus and its separation method and application Download PDFInfo
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Abstract
The present invention relates to one plant of biological and ecological methods to prevent plant disease, pests, and erosion Bei Laisi bacillus and its separation method and applications.The Bei Laisi bacillus strain is on July 25th, 2018 in China typical culture collection center preservation, preservation title are as follows: bacillus 504 or Bacillus velezensis 504, deposit number are CCTCC NO:M 2018493.Bei Laisi bacillus of the invention has significant inhibitory effect to rice leaf spot bacteria, the bacteriostatic activity of wide spectrum is also showed xanthomonas oryzae pv. oryzicola, Ralstonia solanacearum, capsicum A. mali, walnut bacterial black rot bacterium and cowpea wilt etc. simultaneously, and there is important biological and ecological methods to prevent plant disease, pests, and erosion application prospect.
Description
Technical field
The invention belongs to microbial technique application fields, more particularly, to one plant of Bei Laisi bacillus and its separation method
With application.
Background technique
Bacterial blight of rice (Bacterial Blight, BB) and bacterial leaf streak of rice (Bacterial Leaf
Streak, BLS) it is significant bacterial disease common in Rice Production, it is by rice leaf spot bacteria (Xanthomonas respectively
Oryzae pv.oryzae, Xoo) and Xanthomonas oryzae pv. oryzicola (X.oryzae pv.oryzicola, Xoc) caused by (Zou Lifang,
Foundation [J] plant of Zhou Dan, Liu Zhiyang, Zou Huasong, Chen Gong friend's rice Xanthomonas pathogenic related gene insertion mutation system
Pathology journal, 2012,42 (02): 176-185.).Xanthomonas campestris infect and breeding is depended primarily in host tissue endocrine
Polysaccharide, absorption element, virulence factors (Tang Qinghua, Zhang Shiqing, Niu Xiaoqing, Zhu Hui, the Yu Feng such as III type excretory system and lipopolysaccharides
Jade, Qin Wei weigh progress [J] the biological epidemics science of Xanthomonas campestris virulence factor regulation, 2012,35 (02): 134-
141.).Bacterial blight of rice morbidity range is extremely wide, major rice producing region all over the world；Rice is by after bacterial leaf spot disease, edge
Vein generates canescence scab, and rice yield generally reduces 20%~30%, and even have no harvest (Zhai Wenxue, Zhu Li bright water when serious
The research of bacterial blight of rice resistant gene and molecular breeding Chinese biological engineering magazine .1999).Bacterial leaf streak of rice is
A kind of important quarantine bacteriosis in Rice Production, localized epidemics are in southern china and country in Southeast Asia；Rice infects item
It is in streak symptom on blade after pinta, up to 40%~60%, harm is more more than bacterial leaf-blight (Zhang Rong for the rice underproduction when serious
Victory etc., bacterial leaf streak of rice progress, Jiangsu's agriculture journal (JiangsuJ.ofAgr.Sci.), 2014,30 (4):
901-908)。
Currently, mainly including chemical prevention and cultural control to the control method of both rice diseases in production.Chemistry
Prevention and treatment, which exists, the drawbacks such as pollutes environment, endangers human and livestock health and be easy to cause pathogen to be mutated and develop drug resistance；Cultural control
It is difficult to large-scale promotion again.Therefore, biological control is carried out using beneficial microbe have become the main trend (ancestor in agricultural production
English, Zhao Yueju, Liu Yang, mono- plant of Bei Laisi bacillus of Yang Qing benefit inhibit research [J] nuclear agricultural science report of Fusarium graminearum,
2018,32 (02): 310-317.).Hot spot one of of the bacillus as biological control research field, can be by secreting antibacterial
The antibacterial substances such as albumen, lipopeptid class and polyketone class and the growth for inhibiting phytopathogen, to preferably promote plant growth (mulberry
Big, Yang Yang, Chen Yipeng, Cai Jimiao are built, Lu Cuimei, Huang Gui repair raw bacillus amyloliquefaciens BEB17 lipopeptid class and polyketone class in
The bacteriostatic activity for closing object analyzes [J] Plant Pathology, 2018,48 (03): 402-412.).Both environment and people and animals' peace had been ensured
Entirely, it and can effectively prevent pathogen from generating resistance, while meet consumer to agricultural output, quality and safety
Multiple requesting.Therefore, bacillus has great application prospect (Chen Zhiyi, Liu Yongfeng, Liu's postal in modern agricultural production
Continent, Zhang Rongsheng plant disease Biocontrol Bacillus progress [J] Jiangsu's agriculture journal, 2012,28 (05): 999-
1006.)。
Bei Laisi bacillus (Bacillus velezensis) is a novel species for bacillus, is distributed widely in
Various ecological environments (Cai Gaolei, Zhang Fan, Ou Yangyouxiang, Zhao Changsong, Peng Xuanhe, the river such as soil, plant rhizosphere and inside, river
Like the bright north Bei Laisi bacillus (Bacillus velezensis) progress [J] gardening, 2018 (12): 162-
167.).As Biocontrol Bacillus, equally has the function of promoting plant growth and resist pathogenic microorganism, with wide spectrum
Antibacterial activity can be used as the biological control regulator in agricultural production.Bei Laisi bacillus inhibits phytopathogen growth
Mechanism, which specifically includes that, generates antibacterial protein and lipopeptide antibiotic, is synthesized with Nonribosomal Peptide Synthetases and polyketide
Antibiotic and induction plant generation system resistance；By generating IAA, NH3Promote plant growth with acc deaminase.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide one plant of Bei Laisi gemma bars
Bacterium and its separation method and application.
Bei Laisi bacillus of the invention has significant antagonism to rice leaf spot bacteria.
The purpose of the present invention can be achieved through the following technical solutions:
The first aspect of the present invention is: providing one plant of Bei Laisi bacillus, it is empty to be isolated from Sanming City, Fujian Province Youxi County
Heart horticultural vegetable field soil is named as bacillus 504 or Bacillus velezensis 504, and the Bei Laisi bacillus strain is
On July 25th, 2018 in China typical culture collection center preservation, deposit number is CCTCC NO:M 2018493.Preservation
Address are as follows: in the school (the first attached primary school of Wuhan University opposite), Wuhan is big for No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road
Learn collection.
By solid medium tablets culture, the colony colour for observing the Bei Laisi bacillus is milky, edge
Rough, irregularly, dry tack free is coarse, opaque；By micro- sem observation, the thallus of the bacterial strain is rod-short, be can produce
Gemma.
It is tested by Physiology and biochemistry, specifying the Bei Laisi bacillus is one plant of gram-positive bacterium, has and generates
H2The ability of S；Gelatinase can be secreted, beta galactosidase, arginine dihydrolase, lysine decarboxylase, bird cannot be hydrolyzed
Adnosine deaminase decarboxylase, tryptophan deaminase and urase；Acetyl methyl carbinol can be generated using 3-Hydroxybutanone, Yin cannot be generated
Diindyl；Sucrose can be aoxidized, oxidizing glucose, mannitol, inositol, sorbierite, rhamnose, melibiose and amarogentin are unable to.Energy
Enough utilize 17 kinds of carbon sources such as glycerol, glucose, fructose, mannose, inositol, mannitol, sorbierite.
The rice Xanthomonas includes two pvs oryzae and oryzicolas of Xanthomonas oryzae pv. oryzicola Xoc and rice leaf spot bacteria Xoo.
By plate opposite culture method, Bei Laisi bacillus 504 is detected to rice leaf spot bacteria and rice bacterium
The antagonism of property Population of Xanthomonas Oryzae Pv, it was demonstrated that its antagonistic activity that 2 class pathogens are showed with wide spectrum.
Further, the Bei Laisi bacillus is had detected to the antagonism of other 9 kinds of cause of disease Xanthomonas campestris, it was demonstrated that its is right
9 plants of pathogenics such as Ralstonia solanacearum, capsicum A. mali, walnut bacterial black rot bacterium and cowpea wilt
Bacterium also has antagonistic activity.
The second aspect of the present invention is: providing the separation method of the Bei Laisi bacillus: using gradient dilution coated plate
Soil sample plus sterile water dissolution concussion 15min are successively diluted to the soil bacteria suspension of different gradients, are respectively coated on and have connect by method
On the NA plate of kind Xanthomonas oryzae pv. oryzicola, inhibition zone generation has been seen whether, the bacterium colony that will generate obvious inhibition zone is flat in NA
Lining out obtains single colonie, carries out PCR amplification using bacterial 16 S rRNA gene and gyrA gene, and construct phyletic evolution
Tree, specifies the classification position of the bacterial strain.
The present invention constructs systematic evolution tree using 16S rRNA gene and gyrA gene respectively, carries out the comparison of affiliation
Analysis, and combine the physiological and biochemical property of bacterial strain, it was demonstrated that the bacterial strain is Bei Laisi bacillus (Bacillus
velezensis)。
Wherein, the sequence of 16S rRNA gene is as shown in SEQ ID NO.1, the sequence of gyrA gene such as SEQ ID NO.2
It is shown.
The third aspect of the present invention is: the application of the bacillus is provided, including is applied below:
In the specific embodiment of the present invention, the Bei Laisi bacillus 504 to Xanthomonas oryzae pv. oryzicola Xoc and
Rice leaf spot bacteria Xoo all has the antagonism of wide spectrum.
In the specific embodiment of the present invention, the Bei Laisi bacillus 504 has rice leaf spot bacteria
There is significant antagonism.
In the specific embodiment of the present invention, the rice leaf spot bacteria include 8569, YC2, AH1, YC6,
YC11、YN04-1、LYG46、JL3、PXO99A、YC18、XZ35、YC7。
In the specific embodiment of the present invention, the Bei Laisi bacillus 504 has Xanthomonas oryzae pv. oryzicola
Antagonism.
In the specific embodiment of the present invention, the Xanthomonas oryzae pv. oryzicola include HNB07-3, RS105, RS85,
HNB3-17、HANB12-26、ZJB01-25、HANB1-19、JSB1-39、AHB3-7、HNB8-47、AHB1-58、YNB01-3。
In the specific embodiment of the present invention, the Bei Laisi bacillus 504 a variety of yellow unit cells are belonged to its
His plant pathogenetic bacteria, including Ralstonia solanacearum (X.campestris pv.musacearum), capsicum spot disease
Bacterium (X.campestris pv.vesicatoria), walnut bacterial black rot bacterium (X.campestris
Pv.Juglandis), cowpea wilt (X.axonopodis pv.vignicola), Kidney bean wilt
(X.campestris pv.phaseoli), onion Bacterial Leaf Blight bacterium (X.axonopodis pv.allii), tomato blueness are withered
Germ (Ralstonia solanacearum), cobb's disease bacterium (X.axonopodis pv.vasculorum), cotton are thin
Bacterium property angular leaf spot fungus (X.campestris pv.malvacearum).
Compared with prior art, one plant of Bei Laisi bacillus provided by the invention, to rice leaf spot bacteria and rice
Population of Xanthomonas Oryzae Pv all has significant antagonism, and also has to the pathogenetic bacteria of a variety of xanthomonas common at present short of money
Anti- effect is a kind of comparatively ideal Biocontrol microorganism resource, before the field of biological control of plant disease shows greatly application
Scape.Biological control for the various plants bacteriosis in agricultural production now provides new resources.
Detailed description of the invention
Microscope (1000X) the observation photo and colonial morphology of Fig. 1 Bei Laisi bacillus 504.
The phylogenetic tree gene constructed based on 16S rRNA of Fig. 2 Bei Laisi bacillus 504.
The phylogenetic tree gene constructed based on gyr A of Fig. 3 Bei Laisi bacillus 504.
The gel electrophoresis result of Fig. 4 .16S rRNA gene.Wherein 1 represent Marker；2 represent the production of 16S rRNA gene
Object.
The gel electrophoresis result of Fig. 5 .gyrA gene.Wherein 1 represent Marker；2 represent the product of gyrA gene.
Fig. 6 Bei Laisi bacillus 504 is to 12 plants of rice leaf spot bacteria (Xanthomonas oryzae
Pv.oryzae, Xoo) antagonistic effect figure, A:8569；B:YC2；C:AH1；D:YC6；E:YC11；F:YN04-1；G:LYG46；
H:JL3；I:PXO99A；J:YC18；K:XZ35；L:YC7.
Fig. 7 Bei Laisi bacillus 504 is to 12 plants of Xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae
Pv.oryzicola, Xoc) antagonistic effect figure, A:HNB07-3；B:RS105；C:RS85；D:HNB3-17；E:HANB12-26；
F:ZJB01-25；G:HANB1-19；H:JSB1-39；I:AHB3-7；J:HNB8-47；K:AHB1-58；L:YNB01-3.
Fig. 8 Bei Laisi bacillus 504 to the antagonistic effect figures of other 9 kinds of pathogenic Xanthomonas campestris and Raul Salmonella,
A: Ralstonia solanacearum (X.campestris pv.musacearum)；B: cowpea wilt (X.axonopodis
pv.vignicola)；C: walnut bacterial black rot bacterium (Xanthomonas campestris pv.juglandis)；D: kind
Solanum solanacearum (Ralstonia solanacearum)；E: onion Bacterial Leaf Blight bacterium (X.axonopodis
pv.allii)；F: capsicum A. mali (X.campestris pv.vesicatoria)；G: cotton bacterial angular leaf spot bacterium
(X.campestris pv.malvacearum)；H: Kidney bean wilt (X.campestris pv.phaseoli)；I: sugarcane
Gummosis germ (X.axonopodis pv.vasculorum).
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.It should be appreciated that described herein, specific examples are only used to explain the present invention, without constituting to the present invention
Limitation.
Bacterium culture medium used in following embodiment is as follows:
Beef-protein medium NA (g/L): beef extract 3g, polyprotein peptone 5g, sucrose 10g, yeast powder 1g, fine jade
Cosmetics 15g is dissolved in water and is settled to 1000mL, adjusts pH 7.0-7.2, high pressure sterilization (121 DEG C, 20min).
The acquisition of embodiment 1, Bei Laisi bacillus 504
1, soil sources
The hollow horticultural vegetable field soil of Sanming City, Fujian Province Youxi County
2, the screening of bacterial strain
(1) soil sample acquires
5 point sampling method of zigzag carries out soil sample acquisition: soil sample is uniformly mixed, is taken with quartering by every acquisition 200g soil sample
200g is fitted into sterilizing bag, as a soil sample.Every piece of ground acquires 3 soil samples as repetition.Record the time sampled, place
And type.The soil sample that acquisition is completed, which is placed in 4 DEG C of refrigerators, to be saved, in case used in bacterium separation.
(2) separation of bacterium
Plate dilution method: weighing 10g soil sample in conical flask, 90mL sterile water is added, then in 200rpm, 28 DEG C of shaking tables
Middle oscillation takes out after 15min and stands 5min at room temperature, soil bacteria suspension stoste is made.Soil bacteria suspension stoste is subjected to ladder
Degree dilution, respectively obtains 10-1、10-2、10-3、10-4、10-5, the bacteria suspension dilution of totally 6 gradients.Respectively take 200 μ L bacteria suspensions
Dilution is spread evenly across on the NA plate containing Xanthomonas oryzae pv. oryzicola RS105, and each gradient does 2~3 repetitions.It is put into 28
In DEG C biochemical cultivation case, culture is put upside down for 24 hours, observation.
(3) bacteria purification
The single colonie with inhibition zone is picked out in observation, scribing line purifying is carried out on NA plate, in 28 DEG C of biochemical cultivation cases
It is inverted culture, picking single colonie after 12h, number consecutively.
(4) preservation of bacterium
By strain inoculated in liquid NA culture medium, in 28 DEG C, 180rpm shaking table cultivate 12h after, draw 1mL bacterium solution with
The sterile glycerol of 1mL 50%, gently oscillation mixes, and is placed in -80 DEG C of long-term preservations.
(5) screening of Antagonistic Fungi
Using Odontothrips loti: pathogen being seeded in NA fluid nutrient medium, 28 DEG C, cultivate 12h in 180rpm shaking table, inhaled
After taking 200 μ L bacteria suspensions and NA solid medium to mix well, then a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices puts the ox that diameter is 8mm in NA plate center
Saliva cup, each Oxford cup be inscribed 10 μ L for try bacillus liquid (OD600About 2.0), every pathogen strain bacterium is cooked 2~3 repetitions, is set
It is cultivated in 28 DEG C of biochemical cultivation case for 24 hours, the presence or absence of observation inhibition zone records strain number and measures inhibition zone size, whole
Reason is taken pictures.
The Antagonistic Fungi effect filtered out it is best be named as Bei Laisi bacillus (Bacillus velezensis) 504,
By solid medium tablets culture, the colony colour for observing the Bei Laisi bacillus is milky, and edge is rough, no
Rule, dry tack free is coarse, opaque；By micro- sem observation, the thallus of the bacterial strain is rod-short, can produce gemma.Bei Lai
Microscope (1000X) the observation photo and colonial morphology of this bacillus 504 are as shown in Figure 1.
The 16S rRNA identified for genes of 2 Bei Laisi bacillus 504 of embodiment
The genomic DNA for extracting bacterial strain 504, utilizes primer: 27F
5'-AGAGTTTGATCCTGGCTCAG-3 ' and 1492R
5 '-TACGGCTACCTTGTTACGACTT-3 ' carry out PCR amplification, using the gDNA of extraction as template to obtain mesh
Segment.PCR reaction system are as follows:
1 Taq polymerase chain reaction system of table
PCR reacts primary condition are as follows: 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 90s
(1kb/min), 72 DEG C of pre- extension 8min, 4 DEG C of preservations, totally 30 recycle.After reaction, PCR product passes through 1% agarose
Gel electrophoresis is examined, and is detected using gel imager and is recorded result (see attached drawing 4).By PCR stoste send platinum still biotechnology (on
Sea) Co., Ltd is sequenced.Sequencing result is analyzed with DNAStar, and BLAST comparison is carried out on the website NCBI,
Determine the kind of nearly edge strain bacterium.
As the result is shown: the 16SrRNA gene of bacterial strain 504 and Bacillus velezensis with 99.78% it is similar
Degree.The phylogenetic tree that pseudogene is held using MEGA6.0 building, is as a result shown in Fig. 2.
The gyrA identified for genes of 3 Bei Laisi bacillus 504 of embodiment
The genomic DNA for extracting bacterial strain 504, utilizes primer: GyrA-F 5'-CAGTCAGGAAATGCGTACGTCCTT-3'
PCR amplification is carried out using the DNA of extraction as template with GyrA-R 5'-CAAGGTAATGCTCCAGGCATTGCT-3', to obtain
Target fragment.PCR reaction system are as follows:
2 Taq polymerase chain reaction system of table
Reaction condition: 94 DEG C of 10min；94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃
10min, 10 DEG C of ∞.After reaction, PCR product passes through the inspection of 1% agarose gel electrophoresis, is detected using gel imager
And record result (see attached drawing 5).PCR stoste Song Boshang biotechnology (Shanghai) Co., Ltd. is sequenced.Sequencing result fortune
It is analyzed with DNAStar, and carries out BLAST comparison on the website NCBI, determine the kind of nearly edge strain bacterium.
As the result is shown: the phase of the gyrA gene of bacterial strain 504 and Bacillus velezensis mode bacterium gyrA gene order
It like degree up to 99%, and is only 96% with the similarity of Bacillus amyloliquefaciens mode bacterium gyrA gene order.
The phylogenetic tree that pseudogene is held using MEGA6.0 building, is as a result shown in Fig. 3.
The Physiology and biochemistry of 4 Bei Laisi bacillus 504 of embodiment is identified
The physiological and biochemical property of Bei Laisi bacillus 504 of the invention are as follows: have and generate H2The ability of S；It can secrete
It is de- cannot to hydrolyze beta galactosidase, arginine dihydrolase, lysine decarboxylase, bird adnosine deaminase decarboxylase, tryptophan for gelatinase
Adnosine deaminase and urase；Acetyl methyl carbinol can be generated using 3-Hydroxybutanone, indoles cannot be generated；Sucrose can be aoxidized, it cannot
Oxidizing glucose, mannitol, inositol, sorbierite, rhamnose, melibiose and amarogentin.Glycerol, glucose, fruit can be utilized
17 kinds of carbon sources such as sugar, mannose, inositol, mannitol, sorbierite, are shown in Table 3 and table 4.
3 504 physio-biochemical characteristics of bacterial strain of table-enzyme activity, carbon source oxidation
+: positive reaction；: negative reaction；
4 bacterial strain of table, 504 physio-biochemical characteristics-produce acid using carbon source
+: positive reaction；: negative reaction；W: weakly positive reaction
The antagonism spectrum measurement of embodiment 5, Bei Laisi bacillus 504
1) Bei Laisi bacillus 504 measures the antagonistic activity of 12 kinds of rice leaf spot bacterias
It is inoculated in NA liquid respectively by 12 kinds of different rice rice leaf spot bacterias and for examination Bei Laisi bacillus 504
In culture medium, 28 DEG C, cultivate 12h in 180rpm shaking table after, unified OD600It is 2.0；The corresponding pathogen of 200 μ L is drawn respectively
After liquid and NA solid medium mix well, then a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices puts the Oxford cup that diameter is 8mm, Mei Geniu in NA plate center
Saliva cup be inscribed 10 μ L for trying bacterium, each pathogen cooked 2~3 repetitions, is placed in 28 DEG C of biochemical cultivation case, after culture for 24 hours
The presence or absence of inhibition zone is observed, and records inhibition zone size, arrangement is taken pictures.
Bei Laisi bacillus 504 to 12 plants of rice leaf spot bacterias (Xanthomonas oryzae pv.oryzae,
Xoo antagonistic effect) is as shown in fig. 6, in Fig. 6, A:8569；B:YC2；C:AH1；D:YC6；E:YC11；F:YN04-1；G:
LYG46；H:JL3；I:PXO99A；J:YC18；K:XZ35；L:YC7.The data of corresponding fungistatic effect are as shown in table 5.
The fungistatic effect of 504 pairs of 5 Bei Laisi bacillus of table different rice leaf spot bacterias
2) Bei Laisi bacillus 504 measures the antagonistic activity of 12 kinds of Xanthomonas oryzae pv. oryzicolas
It is inoculated in NA fluid nutrient medium respectively by 12 kinds of different Xanthomonas oryzae pv. oryzicolas and for examination Bei Laisi bacillus 504
In, 28 DEG C, cultivate 12h in 180rpm shaking table after, unified OD600It is 2.0；The corresponding cause of disease bacterium solution of 200 μ L and NA are drawn respectively
After solid medium mixes well, then a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices puts diameter for the Oxford cup of 8mm, in each Oxford cup in NA plate center
Connect 10 μ L for trying bacterium, each pathogen cooked 2~3 repetitions, is placed in 28 DEG C of biochemical cultivation case, culture observation suppression afterwards for 24 hours
The presence or absence of bacterium circle, and inhibition zone size is recorded, arrangement is taken pictures.
Bei Laisi bacillus 504 to 12 plants of Xanthomonas oryzae pv. oryzicolas (Xanthomonas oryzae pv.oryzicola,
Xoc antagonistic effect) is as shown in fig. 7, A:HNB07-3 in Fig. 7；B:RS105；C:RS85；D:HNB3-17；E:HANB12-26；
F:ZJB01-25；G:HANB1-19；H:JSB1-39；I:AHB3-7；J:HNB8-47；K:AHB1-58；L:YNB01-3.Accordingly
The data of fungistatic effect are as shown in table 6.
The fungistatic effect of 504 pairs of 6 Bei Laisi bacillus of table different Xanthomonas oryzae pv. oryzicolas
3) Bei Laisi bacillus 504 measures the antagonistic activity of 9 kinds of phytopathogens
By 9 kinds of pathogens such as Ralstonia solanacearum, walnut alternaria, ralstonia solanacearum of tomato and for trying Bei Laisi
Bacillus 504 is inoculated in respectively in NA fluid nutrient medium, 28 DEG C, cultivate 12h in 180rpm shaking table after, unified OD600It is
2.0；Draw the corresponding cause of disease bacterium solution of 200 μ L respectively and after NA solid medium mixes well, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, then in NA plate center
Put the Oxford cup that diameter is 8mm, each Oxford cup be inscribed 10 μ L for trying bacterium, each pathogen cooked 2~3 repetitions, is placed in
In 28 DEG C of biochemical cultivation case, the presence or absence of inhibition zone is observed in culture afterwards for 24 hours, and records inhibition zone size, and arrangement is taken pictures.
Bei Laisi bacillus 504 to the antagonistic effects of 8 kinds of pathogenic Xanthomonas campestris and Raul Salmonella as shown in figure 8,
In Fig. 8, A: Ralstonia solanacearum (X.campestris pv.musacearum)；B: cowpea wilt
(X.axonopodis pv.vignicola)；C: walnut bacterial black rot bacterium (Xanthomonas campestris
pv.Juglandis)；D: ralstonia solanacearum of tomato (Ralstonia solanacearum)；E: onion Bacterial Leaf Blight bacterium
(X.axonopodis pv.allii)；F: capsicum A. mali (X.campestris pv.vesicatoria)；G: cotton is thin
Bacterium property angular leaf spot fungus (X.campestris pv.malvacearum)；H: Kidney bean wilt (X.campestris
pv.phaseoli)；I: cobb's disease bacterium (X.axonopodis pv.vasculorum).The data of corresponding fungistatic effect
As shown in table 7.
Fungistatic effect of the 7 Bei Laisi bacillus 504 of table to 9 kinds of pathogenetic bacterias
One plant of Bei Laisi bacillus 504 provided by the invention is utilized as a result, is had to rice leaf spot bacteria Xoo aobvious
The antagonism of work has the fungistatic effect of wide spectrum to Xanthomonas oryzae pv. oryzicola Xoc, and thin to the cause of disease of a variety of xanthomonas
Bacterium also has antagonism, and the biological control for rice bacteriosis provides new resources.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Sequence table
<110>Shanghai Communications University
<120>one plants of Bei Laisi bacillus and its separation method and applications
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1441
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensis)
<400> 1
gtgctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg ttagcggcgg 60
acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccggg 120
gctaataccg gatggttgtc tgaaccgcat ggttcagaca taaaaggtgg cttcggctac 180
cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360
gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg 420
ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc taactacgtg 480
ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 540
ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600
ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720
gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtgca cgccgtaaac 780
gatgagtgct aagtgttagg gggtttccgc ccccttagtg ctgcagctaa cgcattaagc 840
actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca 900
caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960
atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac aggtggtgca 1020
tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080
tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga 1140
ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1200
caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac aaatctgttc 1260
tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg 1320
atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 1380
gagtttgtaa cacccgaagt cggtgaggta acctttatgg agccagccgc cgaagttgac 1440
c 1441
<210> 1
<211> 931
<212> DNA
<213>Bei Laisi bacillus (Bacillus velezensis)
<400> 1
ccgtcaacga gcgccagcag gttgattccg aaagacgtct gcagggccgt ttgtttgtac 60
aggttattca aaatgacgtg agcattggcg tcacggcgga tctcaatgac gattctcatt 120
ccgttacggt cggattcgtc tcgcagatcg gtaattcctt cgattttttt gtcccggaca 180
agatccgcga ttttttcaat taatctcgct ttgttcacct gataaggaag ttccgtgaca 240
ataattcttt cttttcccga tgaagtctct tcgatttcag ccttagcccg gatcgtgatt 300
gatccccgtc cggattcata tgccttgcgg atgccgctcc ggcccaaaat ctgacctgca 360
gtcggaaaat ccgggcccgg gatgtattcc atcagctcct ggtttgtaat ctcaggattc 420
tcacttacgg caagcacgcc ttcaatgact tccccaagct gatgcggggg aatgtttgtc 480
gccattccga ccgcaatacc ggcagccccg tttacgagca gattcggaaa tctcgaaggc 540
atgacggcag gctctctttc tgaaccgtca tagttatctt gatagtcaat cgtgtctttc 600
gtaatgtcac gcagaatttc cattgcgatt tttgacattc tcgcttctgt gtaacgcatc 660
gcggccgctg agtcgccgtc aaccgaaccg aagttgccgt gtccgtcaac aagcatgtag 720
cggtagttaa aatcctgcgc cattctgacc attgattcgt aaaccgctga gtcaccgtgc 780
gggtggtact taccgataac ttcaccgacg atacgggcag attttttata tggtttgtca 840
ctggtcatgc ctaaatcatt cattgcgtac aaaatccgtc tgtgaaccgg cttcagaccg 900
tcacgcacat ccggaagcgc ccgggatacg a 931
Claims (10)
1. one plant of Bei Laisi bacillus, which is characterized in that the Bei Laisi bacillus strain is on July 25th, 2018 in
State's Type Tissue Collection preservation, deposit number are CCTCC NO:M 2018493.
2. Bei Laisi bacillus according to claim 1, which is characterized in that the Bei Laisi Bacillus colonies are milky white
Color, edge is rough, and irregularly, dry tack free is coarse, opaque；Thallus is rod-short, can produce gemma.
3. Bei Laisi bacillus according to claim 1, which is characterized in that the Bei Laisi bacillus is that a kind of leather is blue
Family name's positive bacteria, rod-short can generate gemma, aerobic or facultative anaerobic bacteria；With generation H2The ability of S；Gelatin can be secreted
Enzyme cannot hydrolyze beta galactosidase, arginine dihydrolase, lysine decarboxylase, bird adnosine deaminase decarboxylase, tryptophan deaminase
And urase；Acetyl methyl carbinol can be generated using 3-Hydroxybutanone, indoles cannot be generated；Sucrose can be aoxidized, cannot be aoxidized
Glucose, mannitol, inositol, sorbierite, rhamnose, melibiose and amarogentin, can be using including glycerol, glucose, fruit
Carbon source including sugar, mannose, inositol, mannitol, sorbierite.
4. Bei Laisi bacillus according to claim 1, which is characterized in that the Bei Laisi bacillus is yellow to rice single
Born of the same parents bacterium has antagonism.
5. Bei Laisi bacillus according to claim 4, which is characterized in that the rice Xanthomonas includes rice streak
Germ (Xanthomonas oryzae pv.oryzicola, Xoc) and rice leaf spot bacteria (Xanthomonas oryzae
Pv.oryzae, Xoo) two pvs oryzae and oryzicolas.
6. Bei Laisi bacillus according to claim 1, which is characterized in that the Bei Laisi bacillus is to the white leaf of rice
Blight bacterium and Xanthomonas oryzae pv. oryzicola have antagonism.
7. Bei Laisi bacillus according to claim 1, which is characterized in that the Bei Laisi bacillus is to pathogenic
Xanthomonas campestris has antagonistic activity.
8. Bei Laisi bacillus according to claim 7, which is characterized in that the pathogenic Xanthomonas campestris includes banana
Bacterial wilt germ, capsicum A. mali, walnut bacterial black rot bacterium and cowpea wilt.
9. the separation method of Bei Laisi bacillus as described in claim 1, which comprises the following steps:
The soil bacteria suspension of different gradients is coated on the NA plate of Inoculated Rice Population of Xanthomonas Oryzae Pv using dilution coated plate method,
Picking can generate the bacterium colony of obvious inhibition zone, and scribing line obtains single colonie, using bacterial 16 S rRNA gene universal primer into
Row PCR amplification constructs systematic evolution tree, specifies the classification position of the bacterial strain.
10. the application of Bei Laisi bacillus as described in claim 1, which is characterized in that including applying below:
Application as rice leaf spot bacteria Antagonistic Fungi；
Application as xanthomonas oryzae pv. oryzicola Antagonistic Fungi；
Application as pathogenic Xanthomonas campestris Antagonistic Fungi；
The pathogenic Xanthomonas campestris includes Ralstonia solanacearum, capsicum A. mali, walnut bacterial black rot
Bacterium and cowpea wilt.
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| CN109929866A (en) * | 2019-02-15 | 2019-06-25 | 黄勇 | The biocontrol bacterial strain Nei Shengbeilaisi bacillus of one plant of tobacco weather-fleck |
| CN110184304A (en) * | 2019-03-27 | 2019-08-30 | 海南大学 | A kind of bacillus HN-2 compound killing pathogenetic bacteria and preparation method |
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| CN107446847A (en) * | 2017-08-14 | 2017-12-08 | 云南农业大学 | One plant of Bei Laisi bacillus GT11 and its application |
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| CN107446847A (en) * | 2017-08-14 | 2017-12-08 | 云南农业大学 | One plant of Bei Laisi bacillus GT11 and its application |
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| CN111254093B (en) * | 2020-01-20 | 2022-01-18 | 上海交通大学 | Bacillus belgii 229-15 and application thereof |
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