CN107779420A - A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application - Google Patents
A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application Download PDFInfo
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- CN107779420A CN107779420A CN201710502641.0A CN201710502641A CN107779420A CN 107779420 A CN107779420 A CN 107779420A CN 201710502641 A CN201710502641 A CN 201710502641A CN 107779420 A CN107779420 A CN 107779420A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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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
Abstract
The present invention relates to a kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application, by in the root of the healthy cigarette strain in the tobacco bacterial wilt diseased region of Enshi separate, screen, obtain raw bacillus R 3 and R 9 in two plants of biological and ecological methods to prevent plant disease, pests, and erosions after purification, two plants of bacterial strains have obvious antagonism to Ralstonia solanacearum, and can produce resistant to elevated temperatures resistance to bacterial wilt fungus matter.Identified by gyrB gene orders, and phylogenetic tree construction, it is Bei Laisi bacillus (Bacillus velezensis) as a result to show R 3, R 9；Pass through potted plant pouring root experimental verification：Bacillus R 3 and R 9 has good colonization ability in Tobacco Root, and can be conveyed into by root in stem；Verified by field experiment：Bei Laisi bacillus R 3 and R 9 prevents and treats tobacco bacterial wilt and worked well, and is significantly better than chemical agent kasugarnycin, and pollution-free.Bacillus R 3 and R 9 research further enriches the research field of biological and ecological methods to prevent plant disease, pests, and erosion endophyte, and the preventing and treating to tobacco bacterial wilt has directive significance.
Description
Technical field
The present invention relates to microbial engineering field, more particularly to a kind of Nei Shengbei of two plants of antagonism tobacco bacterial wilts
This bacillus of Lay and its application.
Background technology
Tobacco bacterial wilt is also known as cigarette pest, is one as caused by Ralstonia solanacearum (Ralstoniasolanacearum)
The native transmissibility bacteriosis of kind, and a kind of typical vascular bundle diseases, most significant disease symptom are withered, once morbidity can make
It is dead into tobacco complete stool, it is one of destructive disease of tobacco, has a strong impact on the yield (Feng Ji etc. 2016) of tobacco leaf.Tobacco green grass or young crops is withered
Disease be particularly acute in Perenniporia martius region, China Yangtze river basin and its on the south cigarette district generally occur.Due to weather
Etc. the reason for, the damaging range of tobacco bacterial wilt was gradually spread from south to north in recent years, and the state of an illness is constantly aggravated, according to 2015
Statistics, the loss of national tobacco is up to 14451.79 ten thousand yuan (old snow etc. 2016) caused by bacterial wilt.
At present, the preventing and treating of tobacco bacterial wilt mainly takes cultural control, biology anti-still without highly effective measure in production
The integrated control policy being combined with chemical prevention is controlled, bacterial wilt onset peak was postponed to the harvesting later stage, so as to reduce loss
(Li Yong and Zhuo Dong 2011).Biological control has the characteristics that economy, safety nuisance free, long-acting, is preventing and treating tobacco bacterial wilt research
Popular direction.The biological control species studied at present mainly includes biological control (Chen Cheng etc. of microbial source and plant source
2011), wherein having turned into the important directions of research using antagonistic microbe controlling plant diseases.
Endophyte of plant is to move in the various tissues and device of health plant in the certain phase of the history of life or whole stages
(Zou Wenxin and Tan appoint auspicious 2001) for fungi or bacterium inside official.Endophyte of plant is a kind of new microbial resources, is had
Very big potential using value.It is the important method of biological control, endophyte and disease using endophyte of plant controlling plant diseases
Opportunistic pathogen has identical ecological niche, vie each other space and nutrition, make pathogen lack nutrition and it is dead, resist disease so as to reach
Harmful purpose (biography of rectifying is rich to wait 2012).In addition, the active material comprising growth-promoting and antibacterial in metabolin caused by endophyte of plant,
Growth and development of plants can be promoted, improve stress resistance of plant, anti-stress, the ability (Xu Ling's tinkling of pieces of jade etc. 2013) of disease resistance.Li Yan the lady in the moons
Deng 7 plants of Antagonistic Endophytics are isolated from the root, stem and leaf of different tobacco breds, wherein B8-5 field efficacies are up to 66.64% (Li Yan
The lady in the moons etc. are 2011).Thin bridge of Peng etc. isolates 32 plants of bacteriums for having antagonism to bacterial wilt from diseased region health tobacco, wherein
H-1, H-9 and H-11 have stronger antagonism, and Preliminary Identification is bacillus (thin bridge of Peng etc. 2007).Xia Yan etc. is from cigarette
3 plants of antagonistic bacteriums for having obvious inhibiting effect to tobacco bacterial wilt, identified 3 plants of antagonisms are isolated and purified out in strain and its Rhizosphere Soil
Bacterium is bacillus, can produce siderophore and indole-3-acetic acid (IAA), and can remarkably promote cigarette strain growth
And reduce the incidence of disease (Xia Yan etc. 2014).
Endophyte is increasingly paid attention to its unique endogeny in biological control, and endophyte is widely present in plant
In object, its theoretical research and Development volue have been to be concerned by more and more people, and have vital meaning in biological control
Justice.
The content of the invention
The technical problems to be solved by the invention are that tobacco bacterial wilt antagonistic activity material can be produced by providing one kind, and
The tobacco endophyte colonized that can be good in tobacco rhizome tissue, the endophyte is environmentally safe, and is easy to industrial metaplasia
Production.
The present invention provides a kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application, its feature exist
Endophyte is isolated in healthy cigarette strain rhizome from bacterial wilt diseased region, 2 plants of experiment acquisition is stood facing each other to green grass or young crops using flat board
Rot has the bacterial strain of bacillus of obvious antagonism, is named as R-3 and R-9, and the zymotic fluid of 2 plants of bacillus is entered
Bacteriostatic test plate is carried out after 115 DEG C of sterilization treatment 10min of row, finds the zymotic fluid after 2 plants of bacillus high-temperature process to green grass or young crops
Withered Lei Er Salmonellas have antagonism；Identify that 2 plants of bacillus are shellfish through gyrB gene sequencings and phylogenetic tree construction
This bacillus (Bacillus velezensis) of Lay；It was preserved on 2 20th, 2017 in China typical culture collection
The heart, preservation address：Wuhan, China.Preserving number is CCTCC NO:M2017050 and CCTCC NO:M2017051；Described one kind
The Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its Bei Laisi bacillus R-3 of application gyrB gene sequences
It is classified as：
GTCTGTCGTAAACGCCTTGTCGACCACTCTTGACGTTACGGTTCATCG
TGACGGAAAAATCCACTATCAGGCGTACGAGCGCGGTGTACCTGTGG
CCGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTACG
CACTTCGTTCCGGATCCGGAAATTTTCAAAGAAACAACCGAATACGA
CTATGACCTGCTTTCAAACCGTGTCCGGGAATTGGCCTTCCTGACAAA
AGGTGTAAACATCACGATTGAAGACAAACGTGAAGGACAAGAACGG
AAAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTATGTTGAGT
ACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGATTTATATC
GAAGGCGAGAAAGACGGCATAACGGTTGAAGTTGCATTGCAATACA
ACGACAGCTATACAAGCAATATTTATTCTTTCACAAATAATATCAACA
CATACGAAGGCGGCACGCACGAAGCCGGATTTAAAACCGGTCTGACC
CGTGTTATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAAA
TGATCCGAATTTAAGCGGGGATGATGTGAGGGAAGGGCTGACTGCCA
TTATTTCAATTAAGCACCCTGATCCGCAATTCGAAGGGCAGACGAAA
ACGAAGCTCGGCAACTCCGAAGCGAGAACGATCACTGATACGCTGTT
TTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCAGCCCG
CAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCA
GCGAAAAAAGCGCGGGAATTGACCCGCCGCAAAAGTGCGCTTGAGA
TTTCCAATCTGCCGGGCAAACTGGCGGACTGTTCTTCTAAAGATCCGA
GCATTTCCGAGCTGTATATCGTAGAGGGTGACTCTGCGGGCGGATCA
GCGAAACAGGGACGGGACCGTCATTTCCAAGCCATTCTGCCGCTGCG
CGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAGATTCTCT
CAAACAATGAGGTCAGATCAATGATCACGGCCCTCGGAACAGGAATC GGAGAAG
Bei Laisi bacillus R-9 GYRB gene orders are：
GCGTCTGTCGTAAACGCCTTGTCGACCACTCTTGACGTTACGGTTCAT
CGTGACGGAAAAATCCACTATCAGGCGTACGAGCGCGGTGTACCTGT
GGCCGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTA
CGCACTTCGTTCCGGATCCGGAAATTTTCAAAGAAACAACCGAATAC
GACTATGACCTGCTTTCAAACCGTGTCCGGGAATTGGCCTTCCTGACA
AAAGGTGTAAACATCACGATTGAAGACAAACGTGAAGGACAAGAAC
GGAAAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTATGTTGA
GTACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGATTTATA
TCGAAGGCGAGAAAGACGGCATAACGGTTGAAGTTGCATTGCAATAC
AACGACAGCTATACAAGCAATATTTATTCTTTCACAAATAATATCAAC
ACATACGAAGGCGGCACGCACGAAGCCGGATTTAAAACCGGTCTGAC
CCGTGTTATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAA
ATGATCCGAATTTAAGCGGGGATGATGTGAGGGAAGGGCTGACTGCC
ATTATTTCAATTAAGCACCCTGATCCGCAATTCGAAGGGCAGACGAA
AACGAAGCTCGGCAACTCCGAAGCGAGAACGATCACTGATACGCTGT
TTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCAGCCC
GCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGC
AGCGAAAAAAGCGCGGGAATTGACCCGCCGCAAAAGTGCGCTTGAG
ATTTCCAATCTGCCGGGCAAACTGGCGGACTGTTCTTCTAAAGATCCG
AGCATTTCCGAGCTGTATATCGTAGAGGGTGACTCTGCGGGCGGATC
AGCGAAACAGGGACGGGACCGTCATTTCCAAGCCATTCTGCCGCTGC
GCGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAGATTCTC
TCAAACAATGAGGTCAGATCAATGATCACGGCCCTCGGAACAGGAAT CGGAGAAGA
A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts of the present invention and its endophyte of application, by bacterium
After strain solid fermentation culture, zymotic fluid is obtained, pouring tobacco using the zymotic fluid of bacterial strain can be in tobacco plant root, stem in 60 days
In colonize, and colonize biomass and be maintained at 103-106cfu/g.
A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts of the present invention and its endophyte of application, can be produced
A kind of raw active material of resistant to elevated temperatures anti-Ralstonia solanacearum, the active material still have anti-Ralstonia solanacearum after handling 10min at 115 DEG C
Activity.
A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts of the present invention and its endophyte of application, with 2
The zymotic fluid of strain bacterial strain dilutes 10 times, pours in the cigarette strain rhizosphere soil of field, each 100 plants of tobaccos of cell, enters after two months
Row Disease investigation；Cigarette strain is poured as medicament control and blank control using 4% kasugarnycin and clear water respectively；Each processing is set
3 cells；Bacterial strain R-3 field control effects are 55.72%, and bacterial strain R-9 field control effects are 50.71%, are significantly better than 4%
Kasugarnycin.
Screened by rifampin gradient and obtain the anti-60 μ g/ml rifampin bacterial strains of R-3 and R-9, and carry out potted plant pouring root respectively
Experiment is colonized, detects the concentration of R-3 and R-9 in Tobacco Root and stem in 7d, 14d, 21d, 28d, 45d, 60d respectively.In Tobacco Root
Middle bacterial strain R-3 keeps stable with the biomass higher than 1 × 106cfu/g, and bacterial strain R-9 slightly float up and down, but biomass one by change
It is straight to keep 1 × more than 104cfu/g；Bacterial strain R-3 keeps stable with the biomass higher than 1 × 105cfu/g in tobacco stem, bacterial strain
Downward trend is presented in R-9, but biomass remains at more than 103cfu/g's after 60d.
Verified by field plot trial, bacterial strain R-3 field control effects are up to 55.72%, bacterial strain R-9 field controls effect
Fruit is 50.71%, is significantly better than 4% kasugarnycin that field efficacy is 31.55%, shows that bacterial strain R-3 and R-9 is being prevented and treated
Farm antibiotics is significantly better than on tobacco bacterial wilt, is a kind of dominant strain.
Advantages of the present invention：2 plants of withered thunder Er Shis blue or green to tobacco are isolated in healthy cigarette strain root from bacterial wilt diseased region
Bacterium has the Nei Shengbeilaisi bacillus of obvious antagonism, and 2 plants of endophytes can produce resistant to elevated temperatures antibacterial substance.Two plants
Endophyte is respectively provided with good colonization ability in tobacco plant, and field control effect is significantly better than farm antibiotics, to ring
Border safety nuisance free, has good DEVELOPMENT PROSPECT and application potential, and the preventing and treating to tobacco bacterial wilt has great importance.
Brief description of the drawings
Accompanying drawing 1 is antagonism schematic diagrames of the bacterial strain R-3 and R-9 to Ralstonia solanacearum；
Accompanying drawing 2 is fungistatic effect schematic diagram of bacterial strain R-3 and the R-9 zymotic fluid to Ralstonia solanacearum；
Accompanying drawing 3 is bacterial strain R-3 and R-9 single bacterium colony figure；
Accompanying drawing 4 is bacterial strain R-3 and R-9 brood cell's colored graph；
Accompanying drawing 5 is the phylogenetic tree of bacterial strain R-3 and R-9 according to gyrB gene orders structure；
Accompanying drawing 6 is bacterial strain R-3 and R-9 the colonization ability broken line schematic diagram in Tobacco Root；
Accompanying drawing 7 is bacterial strain R-3 and R-9 the colonization ability broken line schematic diagram in tobacco stem.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the embodiment not limited to this invented.
Embodiment 1：
The screening and identification of Antagonistic Endophytic
1st, the separation of tobacco endophyte
Healthy cigarette strain is selected in falling ill vega from the bacterial wilt of cloud and mist 87, separates the endogenetic bacteria in root, stem.Weigh respectively
0.5g root, stem carries out surface sterilization：75% ethanol 10s-30s, aseptic water washing twice, 0.1% mercuric chloride 1-3min, sterilized water
(Li Yan the lady in the moons etc. 2011) are flushed three times, then, add the grinding of 5ml sterilized waters fully, 10 times of dilution is coated on LB flat boards, in 37 DEG C
48h is cultivated, the different bacteria distribution purifying of picking colony form, -20 DEG C of glycerol tubes of bacterial strain of purifying are preserved.
2nd, the screening of Antagonistic Endophytic
Ralstonia solanacearum is inoculated with LB, 37 DEG C, 180rpm, is incubated overnight, after 10 times of dilution, LB is uniformly sprayed on and puts down
On plate.37 DEG C of culture 24h of picking endophyte single bacterium drop point is connected on flat board, and in triplicate, 37 DEG C are cultivated 48h to each bacterial strain,
Filter out the bacterial strain (Fig. 1) for producing inhibition zone.
3rd, Antagonistic Endophytic zymotic fluid Activity determination
Choosing has the bacterial strain of antibacterial activity, is inoculated with LB, 28 DEG C, 180rpm, cultivates 48h.Zymotic fluid 10000rpm is centrifuged
10min, 115 DEG C of sterilizing 10min of supernatant are taken, 4 DEG C save backup.Ralstonia solanacearum is uniformly sprayed on LB flat boards.It is flat
4 aseptic filter paper pieces are placed on plate, each filter paper adds 10 μ l zymotic fluids, and after 37 DEG C are cultivated 24h, observation whether there is inhibition zone production
Raw (Fig. 2).
4. colony morphology characteristic and its brood cell's dyeing
Bacterial strain R-3 and R-9 thalline is shaft-like, produces brood cell, and brood cell end life (Fig. 4), bacterium colony circle is flat on LB flat boards, middle
Star-shaped fold projection, rough surface, White-opalescent, there is expansion (Fig. 3).
5th, bacterial strain gyrB gene sequencings are identified
The methods of gyrB gene magnifications primer and reaction condition refer to SATOSHI (Yamamoto S and Harayama S
1995).GyrB gene amplification products are sequenced after purification, BLAST comparisons are carried out by ncbi database, it is soft using MEGA5.2
Part builds the phylogenetic tree of Antagonistic Fungi.Identified bacterial strain R-3 and R-9 bacterium are Bei Laisi bacillus (Bacillus
Velezensis) (Fig. 5).
Embodiment 2：
Endophytic antagonistic bacteria colonizes experiment in tobacco plant
1st, the screening of the antagonistic bacterium of anti-rifampin
2 μ g/ml rifampins are added in LB solid mediums, take 100 μ l bacterium solutions to be coated with, 37 DEG C of culture 2-4d, treat bacterium colony
After growth, picking individual colonies are transferred on 5 μ g/ml rifampicin resistance flat board, and resistant clones are screened after culture.Step up
The concentration of rifampin to 10,20,40,60 μ g/ml, finally obtains anti-60 μ g/ml rifampin bacterial strains respectively.Resistant strain is being added
Add continuous passage 3-5 times, the resistance of stable resistance bacterial strain on the LB flat boards of 60 μ g/ml rifampins.Detect rifampicin resistance bacterial strain
To the antagonistic ability of Ralstonia solanacearum, the antibacterial activity resistant strain similar to starting strain is selected to be used to colonize research.
2nd, antagonistic bacterium colonizes experiment in tobacco body
By the antagonistic strain of the 1% anti-rifampin of inoculation in LB, 37 DEG C, 180rpm fermentations 48h.Tobacco transplant is every after 7 days
Seedling irrigates 50ml zymotic fluids, and each bacterium handles 18 cigarette strains, and every 6 cigarette strains are one group, three groups of parallel tests, to irrigate 50ml
The cigarette strain of water is as a control group.Respectively after 7d, 14d, 21d, 28 d, 45d, 60d, antagonistic bacterium in Tobacco Root and stem is detected
Concentration：The Rhizosphere Soil of tobacco root surface is cleaned, surface moisture is dried, weighs 0.5g roots and stem, surface sterilization is carried out to root and stem,
Method is same as above.It is abundant to add the grinding of 5ml sterilized waters, 10 times of grinding fluid dilution, 100 times takes 100 μ l to be coated on 60 μ g/ml profit good fortune
Flat LB flat boards, 37 DEG C of culture 48h, record clump count, in triplicate, calculate the content of antagonistic bacterium in every gram of root, stem：Per g roots
Antagonistic bacterium content=flat-plate bacterial colony number × extension rate × (sterile water quality+root (stem) quality) × 10/ (stem) in (stem)
Quality (remarks：The sterile water qualities of 1ml are 1g).As a result such as Fig. 6, Fig. 7.Bacterial strain R-3 colonization abilities in tobacco rhizome are preferable,
Pour bacteria containing amount in 60 Tiangengs of processing and maintain more than 106cfu/g, bacteria containing amount maintains more than 105cfu/g in stem；Bacterial strain R-9
After pouring 60 days, bacteria containing amount maintains 105cfu/g in Tobacco Root, and bacteria containing amount drops to 103cfu/g in stem.
Embodiment 3：
Carry out field test in Xuanen County green pepper garden cooling down wind village within 2016.Bacterial strain R-3 and R-9 is inoculated in 2.5L hairs by 1%
In ferment culture medium, 37 DEG C, 180rpm fermentation 48h, 10 times are diluted, takes 200ml to pour in the cigarette strain rhizosphere soil of field.It is each small
100 plants of area tobacco, carries out Disease investigation after two months.Cigarette strain is poured using 4% kasugarnycin and clear water respectively to compare as medicament
And blank control.Each processing sets 3 cells.
After processing is poured 2 months, the incidence of each processing bacterial wilt is investigated, and calculates the incidence of disease, disease index, prevent
Control effect.The prevention effect that the results are shown in Table 1, R-3, R-9 is respectively 55.72%, 50.71%, is significantly higher than the anti-of kasugarnycin
Effect.
Field efficacy of the 1 2016 years Endophytic antagonistic bacterias of table to tobacco bacterial wilt
Note：There is significant difference (p between different letters<0.05).
The disease index of bacterial wilt is according to People's Republic of China's tobacco business standard tobacco disease scale and investigation method
Investigation, the incidence of disease, disease index and prevention effect are calculated as follows.
Tobacco bacterial wilt severity Scaling standard (in units of strain)：
0 grade：Complete stool is disease-free；
1 grade：Occasionally there is a chlorisis spot in stem, or sick side half is wilting with lower blade；
3 grades：There is black streak in stem, but is no more than half, or sick side half is wilting to 2/3rds blades；
5 grades：Stem's black streak exceedes half, but does not reach at the top of stem, or sick side 2/3rds is withered with blade
Wither；
7 grades：Stem's black streak is reached at the top of stem, or diseased plant blade is all wilting：
9 grades：Diseased plant is substantially withered.
The incidence of disease=disease plant/investigation plant sum × 100%
Disease index=Σ [(disease index × this grade of strain number)/(highest series × total strain number)] × 100
Prevention effect (%)=(control group disease index-treatment group disease index)/control group disease index × 100.
SEQUENCE LISTING
<110>Hubei Tbacco Company Enshi Branch
<120>A kind of Nei Shengbeilaisi bacillus of two plants of antagonism tobacco bacterial wilts and its application
<130> 2017
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1088
<212> DNA
<213>Bacterial strain of bacillus
<400> 1
gtctgtcgta aacgccttgt cgaccactct tgacgttacg gttcatcgtg acggaaaaat 60
ccactatcag gcgtacgagc gcggtgtacc tgtggccgat cttgaagtga tcggtgatac 120
tgataagacc ggaacgatta cgcacttcgt tccggatccg gaaattttca aagaaacaac 180
cgaatacgac tatgacctgc tttcaaaccg tgtccgggaa ttggccttcc tgacaaaagg 240
tgtaaacatc acgattgaag acaaacgtga aggacaagaa cggaaaaacg agtaccacta 300
cgaaggcgga atcaaaagct atgttgagta cttaaaccgt tccaaagaag tcgttcatga 360
agagccgatt tatatcgaag gcgagaaaga cggcataacg gttgaagttg cattgcaata 420
caacgacagc tatacaagca atatttattc tttcacaaat aatatcaaca catacgaagg 480
cggcacgcac gaagccggat ttaaaaccgg tctgacccgt gttataaacg actatgcaag 540
aagaaaaggg attttcaaag aaaatgatcc gaatttaagc ggggatgatg tgagggaagg 600
gctgactgcc attatttcaa ttaagcaccc tgatccgcaa ttcgaagggc agacgaaaac 660
gaagctcggc aactccgaag cgagaacgat cactgatacg ctgttttctt ctgcgctgga 720
aacattcctt cttgaaaatc cggactcagc ccgcaaaatc gttgaaaaag gtttaatggc 780
cgcaagagcg cggatggcag cgaaaaaagc gcgggaattg acccgccgca aaagtgcgct 840
tgagatttcc aatctgccgg gcaaactggc ggactgttct tctaaagatc cgagcatttc 900
cgagctgtat atcgtagagg gtgactctgc gggcggatca gcgaaacagg gacgggaccg 960
tcatttccaa gccattctgc cgctgcgcgg taagattctg aacgttgaga aagccagact 1020
tgataagatt ctctcaaaca atgaggtcag atcaatgatc acggccctcg gaacaggaat 1080
cggagaag 1088
<210> 2
<211> 1091
<212> DNA
<213>Bacterial strain of bacillus
<400> 2
gcgtctgtcg taaacgcctt gtcgaccact cttgacgtta cggttcatcg tgacggaaaa 60
atccactatc aggcgtacga gcgcggtgta cctgtggccg atcttgaagt gatcggtgat 120
actgataaga ccggaacgat tacgcacttc gttccggatc cggaaatttt caaagaaaca 180
accgaatacg actatgacct gctttcaaac cgtgtccggg aattggcctt cctgacaaaa 240
ggtgtaaaca tcacgattga agacaaacgt gaaggacaag aacggaaaaa cgagtaccac 300
tacgaaggcg gaatcaaaag ctatgttgag tacttaaacc gttccaaaga agtcgttcat 360
gaagagccga tttatatcga aggcgagaaa gacggcataa cggttgaagt tgcattgcaa 420
tacaacgaca gctatacaag caatatttat tctttcacaa ataatatcaa cacatacgaa 480
ggcggcacgc acgaagccgg atttaaaacc ggtctgaccc gtgttataaa cgactatgca 540
agaagaaaag ggattttcaa agaaaatgat ccgaatttaa gcggggatga tgtgagggaa 600
gggctgactg ccattatttc aattaagcac cctgatccgc aattcgaagg gcagacgaaa 660
acgaagctcg gcaactccga agcgagaacg atcactgata cgctgttttc ttctgcgctg 720
gaaacattcc ttcttgaaaa tccggactca gcccgcaaaa tcgttgaaaa aggtttaatg 780
gccgcaagag cgcggatggc agcgaaaaaa gcgcgggaat tgacccgccg caaaagtgcg 840
cttgagattt ccaatctgcc gggcaaactg gcggactgtt cttctaaaga tccgagcatt 900
tccgagctgt atatcgtaga gggtgactct gcgggcggat cagcgaaaca gggacgggac 960
cgtcatttcc aagccattct gccgctgcgc ggtaagattc tgaacgttga gaaagccaga 1020
cttgataaga ttctctcaaa caatgaggtc agatcaatga tcacggccct cggaacagga 1080
atcggagaag a 1091
Claims (4)
1. Nei Shengbeilaisi bacillus and its application of a kind of two plants of antagonism tobacco bacterial wilts, it is characterised in that sent out from bacterial wilt
Endophyte is isolated in healthy cigarette strain rhizome in lesion domain, standing facing each other to test using flat board obtains 2 plants to bacterial wilt with obvious
The bacterial strain of bacillus of antagonism, R-3 and R-9 are named as, and the zymotic fluid of 2 plants of bacillus is carried out at 115 DEG C of sterilizings
Bacteriostatic test plate is carried out after reason 10min, it is found that it is short of money the zymotic fluid after 2 plants of bacillus high-temperature process has to Ralstonia solanacearum
Anti- effect；Identify that 2 plants of bacillus are Bei Laisi bacillus through gyrB gene sequencings and phylogenetic tree construction
(Bacillus velezensis)；China typical culture collection center was preserved on 2 20th, 2017, preserving number is
CCTCC NO:M2017050 and CCTCC NO:M2017051.
2. Nei Shengbeilaisi bacillus and its application according to a kind of two plants of antagonism tobacco bacterial wilts described in claim 1
Endophyte, after bacterial strain solid fermentation culture, zymotic fluid is obtained, pouring tobacco using the zymotic fluid of bacterial strain can be in cigarette in 60 days
Colonized in careless plant root, stem, and colonize biomass and be maintained at 103-106cfu/g。
3. Nei Shengbeilaisi bacillus and its application according to a kind of two plants of antagonism tobacco bacterial wilts described in claim 1
Endophyte, can produce a kind of active material of resistant to elevated temperatures anti-Ralstonia solanacearum, and the active material still has after handling 10min at 115 DEG C
There is the activity of anti-Ralstonia solanacearum.
4. Nei Shengbeilaisi bacillus and its application according to a kind of two plants of antagonism tobacco bacterial wilts described in claim 1
Endophytes, 10 times are diluted with the zymotic fluid of 2 plants of bacterial strains, is poured in the cigarette strain rhizosphere soil of field, each 100 plants of tobaccos of cell,
Disease investigation is carried out after two months；Cigarette strain is poured as medicament control and blank control using 4% kasugarnycin and clear water respectively；Often
Individual processing sets 3 cells；Bacterial strain R-3 field control effects are 55.72%, and bacterial strain R-9 field control effects are 50.71%,
It is significantly better than 4% kasugarnycin.
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