CN109493920A - A kind of method and application of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny - Google Patents
A kind of method and application of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny Download PDFInfo
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- CN109493920A CN109493920A CN201811558786.3A CN201811558786A CN109493920A CN 109493920 A CN109493920 A CN 109493920A CN 201811558786 A CN201811558786 A CN 201811558786A CN 109493920 A CN109493920 A CN 109493920A
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Abstract
The invention discloses the methods and application of a kind of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny, belong to phytopathogen separation identification technology field.The method of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny of the present invention is that the kind for isolating and purifying rear and the pathogen of the stevioside leaf pinta through Pathogenicity is analyzed and identified by morphological observation, molecular biology identification combination phylogenetic evolution；The molecular biology identification and phylogenetic evolution analysis are ITS, GAPDH and CHS-1 genetic fragment of amplification pathogen, after homologous comparison, construct the phylogenetic evolution tree of ITS, GAPDH and CHS-1 genetic fragment respectively.The present invention identifies that the method for STEVIA REBAUDIANA Leaf blotch pathogeny is quick, easy, avoids because of the close bring identification mistake of the height of inter-species；It lays the foundation for identification stevioside leaf pinta, prevention and treatment stevioside leaf pinta and STEVIA REBAUDIANA Leaf spot resistant breeding.
Description
Technical field
The invention belongs to phytopathogens to separate identification technology field, and in particular to a kind of Rapid identification stevioside leaf pinta
The method and application of pathogen.
Background technique
STEVIA REBAUDIANA [Stevia rebaudiana (Bertoni)] is composite family, sweetleaf Chrysanthemum herbaceos perennial.Sweetleaf
Chrysanthemum blade is rich in flavonoids and stevioside glycoside chemical components.Flavone compound has a variety of pharmacology such as antitumor, anti-oxidant
Activity；Steviol glycoside (steviol glycosides) is then a kind of new type natural sweetener, with sugariness is high, heat is low, peace
The features such as Atoxic, is increasingly becoming the hot spot of food and medicine area research exploitation.2017 in Dongtai City, Jiangsu Province town Fu An
Different degrees of tikka is presented in STEVIA REBAUDIANA production base discovery stevioside leaf on piece, and serious plant leaf of falling ill is all withered,
The economic flow rate of STEVIA REBAUDIANA is seriously affected.Accordingly, it is determined that the infective pathogen bacterium of stevioside leaf pinta, to the anti-jig of the disease
It is significant.
Colletotrichum (Colletotrichum) Distribution of fungi is extensive, and host is various, can cause a large amount of industrial crops
Anthracnose, such as corn, beans, strawberry, coffee, capsicum, cucurbit, potato etc..Normal cause harm leaves of plants, flower, fruit, stem and spray,
The anthracnose for causing various crops causes the symptoms such as tikka, leaf are withered, it is rotten to spend, fruit corruption and branch are withered, influences the growth hair of plant
It educates, reduces crop quality, can cause fallen flowers, shedding or even plant dead when serious, lead to crop production reduction；It can also cause harm
Post-harvest fruits often result in fruit rot, influence product quality, lead to serious economic loss.
The analysis of nrDNA ITS (Internal transcribed spacer, ITS) sequence is anthrax
Earliest, most methods is applied in bacterium molecule taxonomic identification.1992, Mills etc. analyzed the glue spore charcoal for infecting tropical fruit (tree)
The difference of subcutaneous ulcer (C.gloeosporioides) bacterial strain ITS1 sequence, Sreenivasaprasad etc. are analyzed from strawberry
The polymorphism of the different anthrax strain ITS1 sequences of (Fragaria × ananassa Duch.).Then, Sherriff etc. is constructed
The ITS2 of 13 kinds of 27 bacterial strains and the adjacent method of large ribosomal subunit (Large subunit, LSU) gene order
(Neighbor-Joining, NJ) phylogenetic tree identifies round spore anthrax (C.orbiculare) aggregate species.Moriwaki etc.
By ITS and large ribosomal subunit gene sequencing, 25 kinds of 236 bacterial strains of Japan are had studied, it is believed that rDNA
Network analysis is the effective tool of anthrax-bacilus taxonomic identification, and 11 will be separated to from 8 kinds of orchid family (Orchidaceae) plants
Strain anthrax-bacilus is accredited as novel species and wins peaceful anthrax (C.boninense).Peng Ren etc. is observed by morphological feature cultural colony and host
Range measures and ITS sequence is combined to analyze, and carries out to 27 plants of anthrax-bacilus on south China Zingiber (Zingiberaceae) crop
Identification, identifies 3 kinds, wherein based on glue spore anthrax and capsicum anthrax (C.capsici).Xu Chengnan etc. is to picking up from Liaoning
Blueberry (Vaccinium spp.) anthrax bacteria of province carries out morphology and ITS sequence analysis, identifies spore anthrax full to the brim and glue spore
Anthrax.
Has relevant report about STEVIA REBAUDIANA disease at present.2002, Zhu Dongshun etc. had found sweet tea in Shandong Province Gaomi area
The Major Diseases occurred in Ye Ju production have damping-off, mosaic virus and leaf spot etc., to the cured leaf quality of STEVIA REBAUDIANA and production
Amount causes different degrees of influence.The disease of Hexi Region of Gansu Province STEVIA REBAUDIANA nursery stage becomes in what is aggravated year by year in recent years
Gesture, samping off, damping-off and leaf spot become nursery stage and are easiest to 3 major diseases occurred, bring greater loss to agricultural production.
Since anthrax-bacilus has the distribution characteristics such as generality and extreme damage, and there is no about causing STEVIA REBAUDIANA tikka at present in China
The report of the anthrax-bacilus Biology identification this respect of disease, therefore, need to invent it is a kind of quickly, it is easy to stevioside leaf pinta and
The method that other cause of diseases are precisely identified.
Summary of the invention
Aiming at the problems existing in the prior art, the object of the present invention is to provide a kind of identification STEVIA REBAUDIANAs quickly, easy
The method of Leaf blotch pathogeny, and be identification stevioside leaf pinta, prevention and treatment stevioside leaf pinta and STEVIA REBAUDIANA Leaf spot resistant breeding
It lays the foundation.
In order to achieve the above object, the technical solution of the present invention is as follows:
A kind of method of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny, steps are as follows:
(1) morphological observation: observation cultivated in PDA culture medium isolate and purify after and the STEVIA REBAUDIANA through Pathogenicity
The colonial morphology of Leaf blotch pathogeny, color, the speed of growth and conidial form, producing method；
(2) molecular biology identification
The genomic DNA for extracting pathogen carries out PCR amplification to ITS, GAPDH and CHS-1 gene order of pathogen,
Escherichia coli are connected in cloning vector and converted after amplified production recovery purifying, and picking positive colony is sequenced, and is used
BioEdit software compares sequencing result and analyzes；
(3) phylogenetic evolution is analyzed
Utilize the phylogenetic tree of MEGA7 software building pathogen ITS, GAPDH and CHS-1 gene order；
Combining form observation, molecular biology identification and phylogenetic evolution analyze and identify the kind of pathogen.
On the basis of above scheme, the primer pair of amplification ITS gene is ITS-1F/ITS-4, amplification in step (2)
The primer pair of GAPDH gene is GDF/GDR, and the primer pair of amplification CHS-1 gene is CHS-79F/CHS-345R；Their sequence
As shown in NO:1~6 sequence table SEQ ID.
On the basis of above scheme, PCR amplification program is as follows in step (2): 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation
30s, 56 DEG C of annealing 30s, 72 DEG C of extension 40s, totally 33 recycle, last 72 DEG C of extensions 10min, 4 DEG C of preservations.
On the basis of above scheme, cloning vector described in step (2) is pEASY-T1；The Escherichia coli are
Trans-T1。
On the basis of above scheme, the stevioside leaf pinta pathogenicbacteria separation purifying and Pathogenicity, step is such as
Under:
A, the separation, purifying of pathogen
The blade of stevioside leaf pinta typical case's scab is selected, clip disease is good for junction, after cleaned disinfection, removes blade table
Face moisture is placed on the PDA plate containing streptomysin, is cultivated 3-5 days under 25 DEG C of dark conditions, bacterium colony to be grown, picking colony point
End mycelia, which moves on new PDA plate, to continue to cultivate；
B, Pathogenicity
Disease-free, the seamless STEVIA REBAUDIANA blade of selection health, carries out surface sterilization with alcohol, using inoculated by hypha block method,
The bacterium separated in (a) is inoculated in disease-free, the seamless STEVIA REBAUDIANA blade of health；Inoculation induces under the conditions of being placed on constant temperature and humidity
Morbidity, observes and records incidence daily；The sick sample for the STEVIA REBAUDIANA blade fallen ill after acquisition inoculation, re-starts pathogen point
From purifying, observes new isolate and whether inoculation bacterium is consistent；Consistent the two is pathogen.
On the basis of above scheme, the processing of cleaning and sterilizing described in step (a) are as follows: first use aseptic water washing 3~5
It is secondary, 2min is handled with 70% alcohol surface sterilization 30s, then with 0.1% mercuric chloride, is finally used rinsed with sterile water 3 times.
On the basis of above scheme, inoculated by hypha block method described in step (b) are as follows: cut along colony edge identical big
Small bacteria cake, mycelia is face-down, avoids main lobe arteries and veins is affixed on healthy disease-free, seamless STEVIA REBAUDIANA vacuum side of blade two sides.
The method of above-mentioned Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny is in identification stevioside leaf pinta, prevention and treatment STEVIA REBAUDIANA tikka
Application in disease and STEVIA REBAUDIANA Leaf spot resistant breeding.
The beneficial effects of the present invention are:
(1) present invention realizes the in-vitro separation and culture of STEVIA REBAUDIANA Leaf blotch pathogeny, promotes the pathogen with DNA
Based on detection method development, also carry out the researchs such as genetic diversity for the pathogen from now on and established necessary basis.
(2) present invention realizes the Rapid identification for causing the anthrax-bacilus of stevioside leaf pinta, has easy to operate, sensitivity
The features such as height, high specificity, and testing result is accurate, objective, quick, to the detection efficiency for improving STEVIA REBAUDIANA Leaf blotch pathogeny
And sensitivity is of great significance.
(3) present invention recycles ITS sequence, GAPDH sequence and CHS-1 sequence to carry out on the basis of Morphological Identification
Molecular biology identification is further verified and is supplemented to Morphological Identification, is avoided because the close bring identification of the height of inter-species is wrong
Accidentally.
Detailed description of the invention
Front and back sides morphological feature of Fig. 1 PX-1 bacterial strain in PDA culture medium；
Front and back sides morphological feature and conidium form of Fig. 2 PX-2 bacterial strain in PDA culture medium；
Fig. 3 isolated strains PX-1 and PX-2 are inoculated with the scab phenotype after STEVIA REBAUDIANA blade；
The PCR amplification electropherogram of ITS, GAPDH and CHS-1 segment of Fig. 4 bacterial strain PX-1 and PX-2；
The systematic evolution tree of the ITS gene of Fig. 5 bacterial strain PX-1 and PX-2；
The systematic evolution tree of the GAPDH gene of Fig. 6 bacterial strain PX-1 and PX-2；
The systematic evolution tree of the CHS-1 gene of Fig. 7 bacterial strain PX-1 and PX-2.
Specific embodiment
Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified
The meaning of understanding.
Combined with specific embodiments below, and referring to the data further detailed description present invention.Following embodiment only be
It illustrates the present invention, rather than limits the scope of the invention in any way.
Embodiment 1
A kind of method of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny, steps are as follows:
(1) separation, purifying of pathogen
In July, 2017 has the plant of scab from Dongtai City, Jiangsu Province, STEVIA REBAUDIANA production base, the town Fu An acquisition blade, cuts
Taking disease to be good for junction size is 0.5cm × 0.5cm, is used aseptic water washing 3~5 times first, with 70% alcohol surface sterilization 30s,
2min is handled with 0.1% mercuric chloride again, is finally used rinsed with sterile water 3 times, blade surface moisture is removed, clamps material with sterilizing tweezers
It is laid on the PDA plate culture medium added with streptomysin (40 μ g/mL), is cultivated 3~5 days under 25 DEG C of dark conditions, Cong Xinchang
It is further isolated and purified on colony edge picking a small amount of mycelium inoculation to new PDA plate out, the morphology for recording bacterium colony is special
Sign；
Sick sample is acquired from Dongtai City, Jiangsu Province, STEVIA REBAUDIANA production base, the town Fu An, composes star 1 blade in morbidity STEVIA REBAUDIANA
On be separated to two plants of anthrax-bacilus, be respectively designated as PX-1 and PX-2.
(2) Pathogenicity of pathogen
Disease-free, the seamless STEVIA REBAUDIANA blade of selection health carries out Isolated leaf inoculation experiment in laboratory.Each bacterial strain
It is that 3 repetitions are tested.It carries out surface sterilization with 70% alcohol to be placed in the culture dish for being covered with moisturizing filter paper, using mycelia block
The bacterium isolated and purified in (1) is connect and cultivates 7d on PDA plate by inocalation method, cuts 3mm × 3mm along colony edge with sterilizing knife
The bacteria cake of size, mycelia is face-down, avoids main lobe arteries and veins is affixed on healthy disease-free, seamless STEVIA REBAUDIANA vacuum side of blade two sides；Simultaneously
The PDA culture medium block of blank is inoculated with as control.Culture dish is sealed after inoculation, and morbidity is induced under the conditions of being placed in 25 DEG C, is seen daily
It examines and records incidence；After the onset of STEVIA REBAUDIANA blade after to be seeded, pathogen is re-started and is isolated and purified, new point of observation
It is whether identical from object and inoculation bacterium.
The pathogenic interpretation of result of pathogen separation object:
Using the method for Isolated leaf inoculation, observation statistics is carried out to blade incidence, analysis result is shown in Fig. 3,2 bacterium
Strain PX-1 and PX-2 can induce STEVIA REBAUDIANA blade to fall ill, and just blade infection court of falling ill is in the water stain shape of chlorisis, and middle and later periods scab is in
Taupe.A small amount of diseased tissues microscopy is scraped from the leaf spot lesion of inoculation morbidity, can be observed and the consistent bacterium of inoculating strain
Silk and conidium.Separating again for pathogen is carried out to incidence tissue, same acquisition is consistent with inoculation disease fungus colonial morphology
Culture, complete Koch's Postulates (Kochpostulates) pathogenic detection.Therefore, being inoculated with bacterial strain used is to cause sweet tea
The pathogen of leaf chrysanthemum leaf spot.
(3) identification of pathogen
A, morphological observation: isolated pathogenic strain is inoculated into PDA culture medium, is placed in black in 25 DEG C of incubators
It after dark culture 8d, cuts that fritter is thin, culture medium with mycelia from colony edge surface, is placed on glass slide, is covered with
Center is carved with 1cm2The scraps of paper of square hole, covered are observed mitogenetic at optical microscopy (Olympus CX41, Japan)
The form of spore and conidiophore is simultaneously taken pictures, and measures conidial size with eyepiece micrometer.Exist in conjunction with pathogen bacterium colony
Form and color feature in PDA culture medium, the type of pathogen is determined with reference to " Fungal identification handbook ".
The morphological observation qualification result of pathogen is analyzed:
Morphological feature Preliminary Identification of the PX-1 and PX-2 bacterial strain in PDA culture medium is as follows:
PX-1: as shown in connection with fig. 1, mycelia initial stage is white fluffy in PDA culture medium, and later period bacterium colony center occurs light
Pink colour, from bacterium colony center to bacterium colony surrounding radiation growth.
PX-2: as shown in connection with fig. 2, after cultivating 5d in PDA culture medium, the pathogen bacterium colony is rounded, and mycelia arrangement is whole
Together, lint shape, mycelia just be white, after fade to canescence or Dark grey.After cultivating 7d, it is mitogenetic that bacterium colony surface generates Chinese red
Spore ball, conidium oblong, conidium surface is smooth, colourless, unit cell, and cylindrical or ellipse, most both ends are blunt
Circle, size are (15~22.6 × 4.0~6.5) μm.
B, molecular biology identification
10d is cultivated by isolated strain inoculated to PDA culture medium, being placed in 25 DEG C of incubators, from culture base table
Face scrapes mycelia, and the rapid grind into powder of liquid nitrogen is added, referring to fungal genomic DNA extracts kit (Solarbio, China)
Operating instruction extracts bacterial strain DNA, and DNA sample is put in -20 DEG C and is saved backup.
The area ITS uses the universal primer of fungi ribosomes internal gene transcribed spacers:
ITS-1F:5 '-CTTGGTCATTTAGAGGAAGTAA-3 ' (SEQ ID NO:1)；
ITS-4:5 '-TCCTCCGCTTATTGATATGC-3 ' (SEQ ID NO:2)；
The area GAPDH (glyceraldehyde 3-phosphate dehydro-genase gene) universal primer:
GDF:5 '-GCCGTCAACGACCCCTTCATTGA-3 ' (SEQ ID NO:3)；
GDR:5 '-GGGTGGAGTCGTACTTGAGCATGT-3 ' (SEQ ID NO:4)；
The area CHS-1 (chitin synthetase gene) universal primer:
CHS-79F:5 '-TGGGGCAAGGATGCTTGGAAGAAG-3 ' (SEQ ID NO:5)；
CHS-345R:5 '-TGGAAGAACCATCTGTGAGAGTTG-3 ' (SEQ ID NO:6)；
PCR amplification is carried out to the genomic DNA of pathogen.
50 μ L:DNA template of PCR reaction system, 2 μ L, each 1 μ L of upstream and downstream primer, 10 × PCRbuffer (Mg2+plus)5μL、
dNTP Mixs(10mmol·L-1) 1 μ L, 1 μ L of Taq archaeal dna polymerase, sterile ddH2O polishing is to 50 μ L；
PCR amplification program is as follows: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 40s, altogether
33 circulations, last 72 DEG C of extensions 10min, 4 DEG C of preservations；
It is connected to after amplified production recovery purifying in cloning vector pEASY-T1 and converts Escherichia coli Trans-T1, picking
Positive colony is sequenced, and is compared and analyzed to sequencing result using BioEdit software；
Clone's interpretation of result of PX-1 and PX-2 bacterial strain ITS segment, GAPDH segment and CHS-1 segment:
As shown in connection with fig. 4, general using the area fungi rDNA-ITS using the genomic DNA of bacterial strain PX-1 and PX-2 as template
Primer I TS-1F and ITS-4 are expanded respectively obtains the ITS of 616bp (SEQ ID NO:7) and 613bp (SEQ ID NO:8) size
Sequence；It is expanded respectively using the area GAPDH universal primer GDF and GDR and obtains 252bp (SEQ ID NO:9) and 280bp (SEQ ID
NO:10) the GAPDH sequence of size；It is expanded respectively using the area CHS-1 universal primer CHS-79F and CHS-345R and obtains 299bp
The CHS-1 sequence of (SEQ ID NO:11) and 299bp (SEQ ID NO:12) size.PCR product sequencing result shows, PX-1 and
It is had differences between ITS and GAPDH sequence between PX-2 bacterial strain.Sequence is subjected to homology search in NCBI GenBank,
PX-1 and PX-2 and colletotrichum (Colletotrichum) fungi have high homology, therefore the fungi being separated to is belonged to
In anthrax-bacilus.
C, phylogenetic evolution is analyzed
ITS, GAPDH and CHS-1 that sequencing obtains are subjected to homology search in GenBank, and reported fungi bacterium
The ITS region sequence of strain carries out tetraploid rice, utilizes ortho position addition method (Neighbor-Joining, NJ) structure in MEGA7 software
Build phylogenetic tree；
ITS, GAPDH and CHS-1 sequence construct phylogenetic tree interpretation of result based on PX-1 and PX-2:
In conjunction with shown in Fig. 5, Fig. 6 and Fig. 7, in the systematic evolution tree constructed based on ITS region sequence, PX-1 bacterial strain with
MG600754 and KP743027 etc. (Colletotrichum sojae and Colletotrichum gloeosporioides) is poly-
For one kind；PX-2 bacterial strain then with MH863841 and MF540883 etc. (Colletotrichum gloeosporioides and
Colletotrichum fructicola) similarity is higher gathers for one kind.GAPDH and CHS-1 region sequence is utilized on this basis
The sequence similarity of the systematic evolution tree of building, discovery PX-1 bacterial strain and MG600865 etc. (Colletotrichum sojae) reaches
To the sequence similarity of 100%, PX-2 bacterial strain and MF540891 and MG657351 etc. (Colletotrichum fructicola)
Reach 100%.Combining form observation, molecular biology identification and phylogenetic evolution analyze and identify as a result, further confirming that
The pathogen PX-1 bacterial strain for causing stevioside leaf pinta is soybean anthrax-bacilus (Colletotrichum sojae), and PX-2 bacterial strain is
Fruit is raw anthrax-bacilus (Colletotrichum fructicola).
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Sequence table
<110>Commercial Crop Inst., Jiangsu Prov. Academy of Agricultural Sciences
<120>a kind of method and application of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny
<130> 2018
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213>artificial sequence (internal transcribed spacer)
<400> 2
tcctccgctt attgatatgc 20
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<211> 23
<212> DNA
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<213>artificial sequence (glyceraldehyde-3-phosphate dehydrogenase)
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gggtggagtc gtacttgagc atgt 24
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<400> 5
tggggcaagg atgcttggaa gaag 24
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<211> 24
<212> DNA
<213>artificial sequence (chitin synthase gene)
<400> 6
tggaagaacc atctgtgaga gttg 24
<210> 7
<211> 616
<212> DNA
<213>soybean anthrax-bacilus (Colletotrichum sojae)
<400> 7
cttggtcatt tagaggaagt aaaagtcgta acaaggtctc cgtagggtga cctgcggagg 60
gatcattatc gagttaccgc tccttataac cctttgtgaa cataccccaa acgttgcctc 120
ggcgggcagc cggagcccag ctccggcgcc cggagccgcc gtctcggcgc gccccacccg 180
ccggcggacc actaaactct atttaaacga cgtctcttct gagtggcaca agcaaataat 240
caaaactttt aacaacggat ctcttggttc tggcatcgat gaagaacgca gcgaaatgcg 300
ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc acattgcgcc 360
cgccagcatt ctggcgggca tgcctgttcg agcgtcattt caaccctcaa gcaccgcttg 420
gcgttggggc cctacggctt ccgtaggccc cgaaatacag tggcggaccc tcccggagcc 480
tcctttgcgt agtaacatac cacctcgcac tgggatccgg agggactcct gccgtaaaac 540
cccccaattt tccaaaggtt gacctcggat caggtaggga tacccgctga acttaagcat 600
atcaataagc ggagga 616
<210> 8
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gccgtcaacg accccttcat tgagaccaag tacgctgtga gtagcacccc tccaagctcg 60
ccgcgatatc acgcccgcca cccctcaatc gcgaacgcca gcttctggct gccgatcaga 120
cgccaaaatc aatcaggctc tgatacagcg agcgattgat ggggccggcg cggcggggtc 180
gaacatagcc tcaatggttt cggttgctga tacgccatcc gcaggcctac atgctcaagt 240
acgactccac cc 252
<210> 9
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tggggcaagg atgcttggaa gaagattgtc gtttgcgtcg tgagcgatgg tcgcgccaag 60
atcaacccga gaacgagagc actcctggcc ggtatgggtg tgtaccaaga gggaattgcc 120
aagcagcagg tcaacggcaa ggacgtcacc gcgcacattt acgagtatac gtctcaggtc 180
ggaatgcaga tcaagaacga cgtcgtcacc ctggtcccca agcagcagcc cgttcagatg 240
ctgttctgct tgaaggagaa gaaccaaaag aagatcaact ctcacagatg gttcttcca 299
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<400> 10
cttggtcatt tagaggaagt aaaagtcgta acaaggtctc cgttggtgaa ccagcggagg 60
gatcattact gagtttacgc tctataaccc tttgtgaaca tacctataac tgttgcttcg 120
gcgggtaggg tctccgcgac cctcccggcc tcccgcctcc gggcgggtcg gcgcccgccg 180
gaggataacc aaactctgat ttaacgacgt ttcttctgag tggtacaagc aaataatcaa 240
aacttttaac aacggatctc ttggttctgg catcgatgaa gaacgcagcg aaatgcgata 300
agtaatgtga attgcagaat tcagtgaatc atcgaatctt tgaacgcgca ttgcgcccgc 360
cagcattctg gcgggcatgc ctgttcgagc gtcatttcaa ccctcaagct ctgcttggtg 420
ttggggccct acagctgatg taggccctca aaggtagtgg cggaccctcc cggagcctcc 480
tttgcgtagt aactttacgt ctcgcactgg gatccggagg gactcttgcc gtaaaacccc 540
ccaattttcc aaaggttgac ctcggatcag gtaggaatac ccgctgaact taagcatatc 600
aataagcgga gga 613
<210> 11
<211> 280
<212> DNA
<213>fruit is raw anthrax-bacilus (Colletotrichum fructicola)
<400> 11
gccgtcaacg accccttcat tgagaccaag tacgctgtga gtatcacccc acctacccct 60
ccaaactcgc catgacttcg catccatcac caccaccacc accgctgtca tctacatctc 120
gccacccgcg tttggtaaat aagaaggccg tcatgaatgg aggccaattg aaaccatggg 180
tcgggacggc cggacacatg ctatcactca tatcagcccc atctgtcgca tttactgact 240
cgctcttcac aggcctacat gctcaagtac gactccaccc 280
<210> 12
<211> 299
<212> DNA
<213>fruit is raw anthrax-bacilus (Colletotrichum fructicola)
<400> 12
tggggcaagg atgcttggaa gaagattgtc gtctgtgttg tcagcgacgg tcgtgccaag 60
atcaacccgc gaacaagagc actgctggcc ggtatgggtg tgtaccagga gggtatcgcg 120
aagcaacagg tcaacagcaa ggatgtcacc gcccacattt acgaatacac gacccaggtc 180
ggcatgacga tcaagaacga cgttgtctcc ttggttccta agcagcagcc cgttcagatg 240
ctgttctgct tgaaggagaa gaaccagaag aagatcaact ctcacagatg gttcttcca 299
Claims (10)
1. a kind of method of Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny, it is characterised in that:
It is analyzed and identified by morphological observation, molecular biology identification combination phylogenetic evolution after isolating and purifying and through pathogenic
The kind of the pathogen of the stevioside leaf pinta of measurement；
The molecular biology identification and phylogenetic evolution analysis are ITS, GAPDH and CHS-1 gene of amplification pathogen
Segment after homologous comparison, constructs the phylogenetic evolution tree of ITS, GAPDH and CHS-1 genetic fragment respectively.
2. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 1, it is characterised in that: steps are as follows:
(1) morphological observation: observation cultivated in PDA culture medium isolate and purify after and the STEVIA REBAUDIANA tikka through Pathogenicity
The colonial morphology of sick pathogen, color, the speed of growth and conidial form, producing method；
(2) molecular biology identification
The genomic DNA for extracting pathogen carries out PCR amplification, amplification to ITS, GAPDH and CHS-1 gene order of pathogen
Escherichia coli are connected in cloning vector and converted after product recovery purifying, and picking positive colony is sequenced, and is used
BioEdit software compares sequencing result and analyzes；
(3) phylogenetic evolution is analyzed
Utilize the phylogenetic tree of MEGA7 software building pathogen ITS, GAPDH and CHS-1 gene order；
Combining form observation, molecular biology identification and phylogenetic evolution analyze and identify the kind of pathogen.
3. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 2, it is characterised in that: in step (2)
The primer pair for expanding ITS gene is ITS-1F/ITS-4, and the primer pair of amplification GAPDH gene is GDF/GDR, expands CHS-1 base
The primer pair of cause is CHS-79F/CHS-345R；Their sequence is as shown in NO:1~6 sequence table SEQ ID.
4. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 3, it is characterised in that: in step (2)
50 μ L:DNA template of PCR reaction system, 2 μ L, each 1 μ L of upstream and downstream primer, 10 × PCR buffer (Mg2+plus)5μL、dNTP
Mixs(10mmol·L-1) 1 μ L, 1 μ L of Taq archaeal dna polymerase, sterile ddH2O polishing is to 50 μ L.
5. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 3, it is characterised in that: in step (2)
PCR amplification program is as follows: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 40s, totally 33 are followed
Ring, last 72 DEG C of extensions 10min, 4 DEG C of preservations.
6. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 2, it is characterised in that: in step (2)
The cloning vector is pEASY-T1；The Escherichia coli are Trans-T1.
7. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 2, it is characterised in that: the STEVIA REBAUDIANA
Leaf blotch pathogeny isolates and purifies and Pathogenicity, and steps are as follows:
A, the separation, purifying of pathogen
The blade of stevioside leaf pinta typical case's scab is selected, clip disease is good for junction, after cleaned disinfection, removes blade surface water
Point, it is placed on the PDA plate containing streptomysin, is cultivated 3-5 days under 25 DEG C of dark conditions, bacterium colony to be grown, picking colony tip bacterium
Silk, which moves on new PDA plate, to be continued to cultivate；
B, Pathogenicity
Disease-free, the seamless STEVIA REBAUDIANA blade of selection health, carries out surface sterilization with alcohol, using inoculated by hypha block method, by (a)
Middle isolated bacterium is inoculated in disease-free, the seamless STEVIA REBAUDIANA blade of health；Inoculation induces morbidity under the conditions of being placed on constant temperature and humidity,
Incidence is observed and recorded daily；The sick sample for the STEVIA REBAUDIANA blade fallen ill after acquisition inoculation, it is pure to re-start pathogenicbacteria separation
Change, observes new isolate and whether inoculation bacterium is consistent；Consistent the two is pathogen.
8. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 7, it is characterised in that: in step (a)
The described cleaning and sterilizing processing are as follows: first use aseptic water washing 3~5 times, with 70% alcohol surface sterilization 30s, then with 0.1% mercuric chloride
2min is handled, is finally used rinsed with sterile water 3 times.
9. the method for Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny according to claim 7, it is characterised in that: in step (b)
The inoculated by hypha block method are as follows: cut the bacteria cake of same size along colony edge, mycelia down, avoid main lobe arteries and veins be affixed on it is strong
Health is disease-free, seamless STEVIA REBAUDIANA vacuum side of blade two sides.
10. the method for any one of the claim 1~9 Rapid identification STEVIA REBAUDIANA Leaf blotch pathogeny is in identification STEVIA REBAUDIANA tikka
Application in disease, prevention and treatment stevioside leaf pinta and STEVIA REBAUDIANA Leaf spot resistant breeding.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734922A (en) * | 2019-10-29 | 2020-01-31 | 吉林大学 | Detection method of Colletotrichum camelliae |
| CN110734921A (en) * | 2019-10-29 | 2020-01-31 | 吉林大学 | Detection method of kinds of anthracnose bacteria Colletotrichum siamense of tea trees |
| CN112195217A (en) * | 2020-10-30 | 2021-01-08 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Method for determining pathogenicity of pathogenic bacteria of weedy rice and identification device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399888A (en) * | 2011-11-28 | 2012-04-04 | 江苏省农业科学院 | Molecular identification method for cochliobolus miyabeanus pathogens |
| CN105400708A (en) * | 2015-12-18 | 2016-03-16 | 东莞市香蕉蔬菜研究所 | Banana wilt composite pathogenic bacteria and preparation method and application thereof |
-
2018
- 2018-12-19 CN CN201811558786.3A patent/CN109493920A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399888A (en) * | 2011-11-28 | 2012-04-04 | 江苏省农业科学院 | Molecular identification method for cochliobolus miyabeanus pathogens |
| CN105400708A (en) * | 2015-12-18 | 2016-03-16 | 东莞市香蕉蔬菜研究所 | Banana wilt composite pathogenic bacteria and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 崔晓霞 等: "甜叶菊褐斑病的病原菌鉴定及MeJA的抗病作用", 《中国农业科学》 * |
| 张佳星: "白术、铁皮石斛和杭白菊6种病害的病原鉴定与防治初探", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 马瑞 等: "海南省温郁金炭疽病的病原鉴定", 《植物保护》 * |
| 马甲强: "苜蓿炭疽病病原学及防治研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734922A (en) * | 2019-10-29 | 2020-01-31 | 吉林大学 | Detection method of Colletotrichum camelliae |
| CN110734921A (en) * | 2019-10-29 | 2020-01-31 | 吉林大学 | Detection method of kinds of anthracnose bacteria Colletotrichum siamense of tea trees |
| CN112195217A (en) * | 2020-10-30 | 2021-01-08 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Method for determining pathogenicity of pathogenic bacteria of weedy rice and identification device |
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