CN107299147A - A kind of how main rod spore bacterium of Rapid identification is to the drug-fast method of fluopyram and special primer pair - Google Patents
A kind of how main rod spore bacterium of Rapid identification is to the drug-fast method of fluopyram and special primer pair Download PDFInfo
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Abstract
The invention discloses a kind of how main rod spore bacterium of Rapid identification to the drug-fast method of fluopyram and special primer pair.Detecting how main rod spore bacterium to the application in fluopyram resistance the invention provides the material for detecting the succinate dehydrogenase B subunit coding gene A1020G sites base in how main rod spore bacterium genome；The A1020G sites are the 1020th in the succinate dehydrogenase B subunit coding genes；The base in the A1020G sites is A or G.The method for the point mutation that the how main rod spore bacterium of detection that the present invention is provided develops immunity to drugs to fluopyram, it can be used for enzyme rapidly and sensitively detecting that to the resistance of fluopyram development trend occurs for the how main rod spore bacterium in field, to adjust the control strategy of cucumber rod spore leaf spot in time, to delay and control drug-fast further development.
Description
Technical field
The invention belongs to technical field of molecular biology, more particularly to how main rod spore bacterium succinate dehydrogenase B subunit genes
SdhB clone and its application in fluopyram Resistance detecting and monitoring, specifically disclose a kind of how main rod spore of Rapid identification
Bacterium is to the drug-fast method of fluopyram and special primer pair.
Background technology
In recent years, how main rod spore bacterium (Corynespora cassiicola (Berk.＆M.A.Curtis) C.T.Wei) is drawn
The cucumber rod spore leaf spot risen rises to a kind of Major Diseases by Minor diseases on China's cucumber production.Field morbidity is general
The underproduction 20%, up to 60%~70% when serious.The disease in 1906 Europe report first, nineteen fifty-seven the U.S. northern Caro
Lai Na states report the generation of the disease, in the 1960s, Qi Peikun etc. reports the generation of the disease in China first.20
The nineties in century, room moral disease that reports such as pure turns into Liaoning Province Wafangdian City dependent territory cucumber Major Diseases.In recent years,
In China Liaoning, Hebei, Shandong, Henan, 19 provinces and cities' diseases such as Gansu have evolved into Major Diseases, are caused to cucumber production
Heavy losses, one of leaf diseases urgently to be resolved hurrily in being produced as dependent territory cucumber.
More main rod spore bacterium, is under the jurisdiction of sac fungus gate seat capsule Gammaproteobacteria lattice spore Zoopagales Bang Bao sections Corynespora, is a kind of hereditary variation
The larger fungi of property.This can infect blade, stalk and the root of plant and the sporangiocyst of nematode and the skin of people, and its host plants
Thing includes 530 kinds of plants of 380 category, including unifacial leaf, dicotyledonous etc..Because the pathogen is a kind of group of height variation
Body, thus using resistant variety carry out Disease management when exist the lasting period it is short the problem of.It is currently preventing and treating that chemical prevention, which turns into,
The mostly important means of the disease.The chemical agent effective to cucumber rod spore leaf spot reported both at home and abroad at present mainly includes
Boscalid, metalaxyl, fluopyram, thiophanate-methyl, Fluoxastrobin, Prochloraz, thiram, iprodione, carbendazim, hundred bacterium
Clearly, pyraclostrobin, Mancozeb, famoxadones and their mixture preparation and systemic Plant elicitors diazosulfide.
Wherein most representational is using fluopyram and Boscalid as the succinate dehydrogenase inhibitors of representative
(Succinate dehydrogenase inhibitors, SDHIs).SDHIs is that bactericide resistance action association (FRAC) presses
Belong to the bactericide of amide-type in the structure newly marked off according to its similar mechanism of action and resistance mechanism.Its action target is cause of disease
Succinate dehydrogenase (succinate dehydrogenase, SDH) in fungal mitochondria respiratory chain, i.e. complexⅱ or amber
Amber acid：Ubiquinone oxide-reductase enzyme (succinate：Ubiquinone oxidoreductase, SQR).Including fluopyram,
Boscalid, pyrrole metsulfovax, totally 18 kinds of bactericide such as carboxin.The series bactericidal agent is due to active height, wide sterilization spectrum in full generation
It is widely used in the preventing and treating of corps diseases in the range of boundary.
At present, existing 14 kinds of plant pathogenic fungis report the resistance generation to the series bactericidal agent, and most of on anti-
Property mechanism report article by the B/C/D subunit bases for causing the drug-fast factor of disease fungus to be locked in succinate dehydrogenase
On the site mutation of cause, wherein drug resistance mechanism research of the how main rod spore bacterium to Boscalid shows leading more for Different resistance levels
Rod spore bacterium, its mechanism is different.For the strain of superelevation antibacterial, mainly occur SdhB-H278Y (CAC-TAC) mutation, for high antibacterial
Strain, mainly occurs SdhB-H278R (CAC-CGC) mutation, and for moderate resistance bacterial strain, resistant mutation includes SdhC-S73P (TCG-
CCG), SdhD-G109V (GGC-GTC), SdhD-S89P (TCC-CCC).
For phytopathogen liquefaction resistance or the traditional biological method of monitoring, including colony growth determination method, spore
Son sprouts determination method etc., is the EC by determining medicament to phytopathogen50To distinguish sensitive and resistant strain, than relatively time-consuming
Arduously.In recent years, with the fast development and progress of nucleic acid correlation molecule detection technique, Protocols in Molecular Biology is anti-in pathogen
The property of medicine is detected and is widely used in monitoring, such as AS-PCR (Allele-specific PCR) and PCR-
RFLP(Restriction Fragment Length Polymorphism).Compared to traditional detection method, the latter is from DNA
Specific PCR or restriction analysis are extracted, time-consuming short, sensitivity is high, detection frequency is 10-5~10-4, it is adapted to detect for low frequency and resists
Property of medicine gene, is the Perfected process of a kind of field drug-fastness early detection or monitoring.
The content of the invention
A purpose of the invention is to provide the succinate dehydrogenase B subunit coding genes detected in how main rod spore bacterium genome
The purposes of the material of A1020G sites base.
The invention provides the succinate dehydrogenase B subunit coding gene A1020G sites alkali in how main rod spore bacterium genome
The material of base is detecting how main rod spore bacterium to the application in fluopyram resistance；
The A1020G sites are the 1020th in the succinate dehydrogenase B subunit coding genes；
The base in the A1020G sites is A or G.
Another object of the present invention is to provide the succinate dehydrogenase B subunits the 280th detected in how main rod spore bacterium genome
The purposes of the material of amino acids residue.
The invention provides detect that how main succinate dehydrogenase B the 280th amino acids of subunit in rod spore bacterium genome are residual
The material of base is detecting how main rod spore bacterium to the application in fluopyram resistance；
The 280th amino acids residue is the succinate dehydrogenase B yldeneaminos acid sequence the 280th；
The 280th amino acids residue is Ile or Val.
3rd purpose of the invention is to provide a kind of side of detection or the how main rod spore bacterium of auxiliary detection to fluopyram resistance
Method.
The method that the present invention is provided, comprises the following steps：Butanedioic acid in detection how main rod spore bacterium to be measured in genome takes off
Hydrogen enzyme B subunit coding gene A1020G sites base, the base in the A1020G sites is A or G；If the base in the site is A,
Then the how main rod spore bacterium is or candidate is sensitive to fluopyram；If the base in the site be G, the how main rod spore bacterium be or
Candidate is resistant to fluopyram.
4th purpose of the invention is to provide a kind of side of detection or the how main rod spore bacterium of auxiliary detection to fluopyram resistance
Method.
The method that the present invention is provided, comprises the following steps：Butanedioic acid in detection how main rod spore bacterium to be measured in genome takes off
Hydrogen enzyme B subunit coding genes coding the 280th, albumen amino acid residue, the 280th amino acids residue be Ile or
Val；If the amino acid residue in the site is Ile, the how main rod spore bacterium is or candidate is sensitive to fluopyram；If the position
The amino acid residue of point is Val, then the how main rod spore bacterium is or candidate is resistant to fluopyram.
5th purpose of the invention is to provide a kind of side of detection or the how main rod spore bacterium of auxiliary detection to fluopyram resistance
Method.
The method that the present invention is provided, comprises the following steps：Butanedioic acid in detection how main rod spore bacterium to be measured in genome takes off
Hydrogen enzyme B subunit coding gene A1020G sites base, the base in the A1020G sites is A or G；The base in the site is G's
How main rod spore bacterium to be measured is to the resistance of fluopyram higher than the how main rod spore bacterium to be measured that the base in the site is A.
5th purpose of the invention is to provide a kind of side of detection or the how main rod spore bacterium of auxiliary detection to fluopyram resistance
Method.
The method that the present invention is provided, comprises the following steps：Butanedioic acid in detection how main rod spore bacterium to be measured in genome takes off
Hydrogen enzyme B subunit coding genes coding albumen the amino acid residue of the 280th, the 280th amino acids residue be Ile or
Val；The amino acid residue in the site is higher than the amino in the site for Val how main rod spore bacterium to be measured to the resistance of fluopyram
Sour residue is the how main rod spore bacterium to be measured of isoleucine.
In the above method,
The albumen of succinate dehydrogenase B subunit coding genes coding in the detection how main rod spore bacterium to be measured in genome
The amino acid residue of the 280th is by detecting that the succinate dehydrogenase B subunits in how main rod spore bacterium to be measured in genome encode base
Because A1020G sites base is realized；
Succinate dehydrogenase B subunit coding gene A1020G sites in the detection how main rod spore bacterium to be measured in genome
1) or 2) base is realized by following method：
1) it is sequenced；
2) with the succinate dehydrogenase B subunit coding gene A1020G sites alkali in the how main rod spore bacterium genome of detection
The material of base expands the how main rod spore bacterium to be measured, detects amplified production, described if the amplified production obtains specific fragment
Succinate dehydrogenase B subunit coding gene A1020G sites base in detection how main rod spore bacterium to be measured in genome is G, if expanding
Volume increase thing does not obtain specific fragment, then the succinate dehydrogenase B subunits in the detection how main rod spore bacterium to be measured in genome are compiled
Code Gene A 1020G sites base is A；
Succinate dehydrogenase B subunit coding gene A1020G sites base in the how main rod spore bacterium genome of the detection
Material includes following A or B：
A the primer pair in the A1020G sites) is expanded；
B) PCR reagent or kit containing the primer pair.
In the above method,
The primer pair in the amplification A1020G sites is as the nucleotides shown in the nucleotides shown in sequence 5 and sequence 6
Composition；
Or the specific fragment size is 250-270bp；
Or the annealing temperature of the amplification is 61.4-68.4 DEG C.
The how main rod spore bacterium of the detection in above-mentioned application or the above method is also this to the material of fluopyram resistance
Invent the scope of protection.
6th purpose of the invention is to provide a kind of succinate dehydrogenase B detected in how main rod spore bacterium to be measured in genome
The method of subunit coding gene A1020G sites base.
The method that the present invention is provided, comprises the following steps：Taken off with the butanedioic acid in the how main rod spore bacterium genome of detection
The material of hydrogen enzyme B subunit coding gene A1020G sites base expands the how main rod spore bacterium to be measured, amplified production is detected, if institute
State amplified production and obtain specific fragment, then the succinate dehydrogenase B subunits in the detection how main rod spore bacterium to be measured in genome
Encoding gene A1020G sites base is G, if amplified production is not obtained in specific fragment, the detection how main rod spore bacterium to be measured
Succinate dehydrogenase B subunit coding gene A1020G sites base in genome is A；
Succinate dehydrogenase B subunit coding gene A1020G sites base in the how main rod spore bacterium genome of the detection
Material includes following A or B：
A the primer pair in the A1020G sites) is expanded；
B) PCR reagent or kit containing the primer pair.
In the above method, the primer pair in the amplification A1020G sites is as the nucleotides shown in sequence 5 and sequence 6
Shown nucleotides composition；
Or the specific fragment size is 250-270bp, the specially 260bp in embodiment, nucleotides sequence is classified as sequence 1
In the 785th to 1044 from 5 ' ends.
Or the annealing temperature of the amplification is 61.4-68.4 DEG C.
The experiment proves that, the present invention provides the detection point that how main rod spore bacterium develops immunity to drugs to fluopyram and dashed forward
The method of change, can be used for enzyme rapidly and sensitively detecting that to the resistance of fluopyram development trend occurs for the how main rod spore bacterium in field,
To adjust the control strategy of cucumber rod spore leaf spot in time, to delay and control drug-fast further development.
Brief description of the drawings
Fig. 1 is to show resistance and sensitive how main rod spore bacteria strain succinate dehydrogenase B subunit genes to fluopyram
SdhB global alignment results.
Fig. 2 is to show resistance and sensitive how main rod spore bacteria strain succinate dehydrogenase B subunits SdhB to fluopyram
Global alignment result.
Fig. 3 is to carry out thermograde PCR amplification electrophoretograms using different AS-PCR primer pairs.
Fig. 4 is primer pair specific detection result.
Fig. 5 is primer pair sensitivity technique result.
Fig. 6 is that primer pair is detecting how main rod spore bacterium to the application in fluopyram resistance.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
" resistant strain " is referred both to fluopyram resistance or to fluopyram EC in text50More than or equal to 25 μ g/mL's
More main rod spore bacteria strain；" sensitive strain " is referred both to fluopyram sensitivity or to fluopyram EC50It is many less than 2 μ g/mL
Main rod spore bacteria strain.
The how main rod spore bacteria strain used in following embodiments：HG15050729-5(CGMCC No.14544)、
HG15050825-6, HG15050731 and HG15050737 are resistant strain；HG14102405-2,HG14102524-4(CGMCC
No.14543), HG15050755-3, HG15041321-2 and HG15041414-2 are sensitive strain.
Above-mentioned bacterial strains HG15050755-3, HG15050729-5 and HG15050825-6 are by this problem group membership in 2015
Pick up from year in LiaoNing, China area；HG15041321-2 and HG15041414-2 are picked up from Shandong Province of China area.HG14102405-
2 and HG14102524-4 is picked up from Hebei China area.Each bacterial strain passes through existing morphology and molecular biology side above
Method is accredited as how main rod spore bacteria strain.Above-mentioned each bacterial strain carries out sensitivity testing by colony growth performance rate method and the resistance to the action of a drug is tested
Card.
Succinate dehydrogenase B subunit genes SdhB and its hair in correspondence protein mutation site in embodiment 1, how main rod spore bacterium
It is existing
First, sensitivity testing of the how main rod spore bacterium to fluopyram
5 plants of sensitive strains HG15050755-3, HG15041321-2, HG15041414-2, HG14102405-2 and
HG14102524-4；4 plants of resistant strains HG15050729-5, HG15050731, HG15050737 and HG15050825-6.
Potato dextrose agar (PDA)：Potato 200g, glucose 20g, agar 15g, distilled water is settled to
1L, 121 DEG C of moist heat sterilization 30min.
1) fluopyram is made into 5 × 10 with acetone4μ g/ml mother liquor.For main rod spore bacterium sensitive strain more than 5 plants and
The sensitivity testing of main rod spore bacterium resistant strain more than 4 plants, 10 μ g/mL, 100 μ g/mL, 500 μ g/ are diluted to by fluopyram
ML, 1000 μ g/mL, 5000 μ g/mL, 10000 μ g/mL and 25000 μ g/mL concentration gradient.
2) by order from low to high, the μ L of decoction 135 additions that each concentration gradient is drawn with liquid-transfering gun are sterilized cold
But into 55 DEG C of 135mL PDA culture mediums, it is 1 ‰ to make solvent, mixes, the culture medium of pastille is poured into a diameter of
In 5.5cm culture dish, only to add the processing of 135 μ L acetone as blank control.Each concentration is repeated 3 times.
3) strains tested is activated from being stored on 4 DEG C of inclined-plane, dark culturing after 5 days at 26 DEG C, with card punch along bacterium colony
The bacteria cake for taking a diameter of 0.5cm is beaten at edge, and mycelia is inoculated in step 2 downwards) in pastille and control flat board in, be placed in 26 DEG C
Colony diameter is measured after dark culturing in incubator, 5d.
4) crossing method measurement colony diameter, is calculated under each concentration to being led for examination more according to bacterium colony mean diameter value
The mycelial growth inhibition rate of rod spore bacteria strain.Then the EC50 values of each bacterial strain are calculated with the probit of SAS 9.4 analyses and its put
Letter is interval.
As a result as shown in table 1, sensitivity testing result shows, fluopyram to sensitive strain HG15050755-3,
HG15041321-2, HG15041414-2, HG14102405-2 and HG14102524-4 EC50Respectively less than 2 μ g/mL, and resist
Property bacterial strain HG15050729-5, HG15050731, HG15050737 and HG15050825-6 EC50It is all higher than 25 μ g/mL, resistance
Multiple is more than 12.5 times.
Table main rod spore bacterium sensitive strain more than 15 plants and sensitiveness of the main rod spore bacterium resistant strain to fluopyram more than 4 plants
2nd, succinate dehydrogenase B subunit genes SdhB and its correspondingly discovery in protein mutation site in how main rod spore bacterium
1st, bacterial strain：Resistant strain HG15050729-5, HG15050731, HG15050737 and HG15050825-6, it is sensitive
Bacterial strain HG15050755-3, HG15041321-2, HG15041414-2, HG14102405-2 and HG14102524-4.
2nd, method：
1) template：Respectively using resistant strain and the genomic DNA of sensitive strain as template.
2) the PCR amplifications of succinate dehydrogenase B subunit genes SdhB complete sequences
Primer (table 2)：CcSdhB-F(5’-CGCAACGACACTCTTCTT-3’)
CcSdhB-R(5’-CCATTCAGCAACGACTCT-3’)
Using the rich Deco skill Development Co., Ltd advanced in years reagent in Beijing, 50 μ L reaction systems include 2 μ L template DNAs (30-
50ng), every primer (10 μM) 2 μ L, 25 μ 2 × Taq of L Mix (purchased from the rich Deco skill Development Co., Ltd advanced in years in Beijing), 19 μ L
ddH2O, reaction system needs to be increased and decreased at double according to follow-up test.Response procedures are：94 DEG C of 5min of pre-degeneration；94 DEG C of 45s,
58 DEG C of 45s, 72 DEG C of 2min, 35 circulations；Extend 5min at last 72 DEG C.Amplified production electrophoresis in 1% Ago-Gel
Detection.
3) it is sequenced
PCR primer is sequenced, sequencing primer is shown in Table 2.During using genomic DNA as template, the butanedioic acid of resistant strain
There are 3 sections of intron sequences, 307 encoded altogether in the dehydrogenase B subunit gene common 1106bp of SdhB complete sequences, sequence as shown in sequence 1
Individual amino acid, sequence is as shown in sequence 2.
Respectively by resistant strain HG15050729-5HG15050731, HG15050737 and HG15050825-6, with sensitivity
Bacterial strain HG15050755-3, HG15041321-2, HG15041414-2 and HG14102405-2 succinate dehydrogenase B subunit bases
Because sdhB complete sequences are sequenced.By DANMAN software analysis sequence results, there is following mutational site in discovery：With sensitive bacteria
Strain is compared, resistant strain HG15050729-5, HG15050731, HG15050737 and HG15050825-6 succinate dehydrogenase
B subunit genes sdhB sports G (Fig. 1) in the 1020th of gene order the nucleotides from 5 ' ends by A, and the site is
A1020G mutational site, the site base mutation causes amino acid sequence to be undergone mutation at the 280th, by isoleucine
(Ile, I) sports valine (Val, V) (Fig. 2).
Amber in above-mentioned resistant strain HG15050729-5, HG15050731, HG15050737 and HG15050825-6
Acidohydrogenase B subunit gene sdhB nucleotides sequences are classified as sequence 1, the amino acid of its albumen succinate dehydrogenase B subunit encoded
Sequence is that the succinate dehydrogenase B subunits in sequence 2, resistant strain are denoted as how main rod spore bacterium succinate dehydrogenase B subunits mutation
Body, is named as SdhB-V280.
Above-mentioned sensitive strain HG15050755-3, HG15041321-2, HG15041414-2, HG14102405-2 and
Succinate dehydrogenase B subunit gene sdhB nucleotides sequences in HG14102524-4 are classified as sequence 3, its albumen butanedioic acid encoded
The amino acid sequence of dehydrogenase B subunits is that the succinate dehydrogenase B subunits in sequence 4, sensitive strain are denoted as how main rod spore bacterium amber
Amber acidohydrogenase B subunits, are named as SdhB-I280.
The 1020th A of sequence 3 is is sported G by sequence 1, the sequence that other invariant nucleotides are obtained；
The 280th I of sequence 4 is is sported V by sequence 2, the constant obtained sequence of other amino acid.
It therefore, it can by detecting how main rod spore bacterium succinate dehydrogenase B subunit genes nucleotide sequence the 1020th
Base is A or G, to detect that how main rod spore bacterium is resistance or sensitive to fluopyram.
Mutational site A1020G method in embodiment 2, the how main rod spore bacterium of detection
First, design of primers, screening and amplification method
Bacterial strain be embodiment 1 in bacterial strain uses therefor HG15050729-5, HG15050825-6, HG14102524-4,
HG14102405-2、HG15041321-2、HG15041414-2、HG15050755-3。
According to mutant HG15050729-5 succinate dehydrogenase B subunit genes SdhB sequences Design AS-PCR primers.
Primer pair Cc1020F-Cc1020A/T/C/G.C.R is used for the mutational site for detecting A1020G.Wherein, reverse primer
The 3 ' of Cc1020A.C.R-hold last base to be the base after mutation, forward primer is Cc1020F (table 2).In order to increase
The specificity of primer, base mismatch (table 2, primer Cc1020T/C/ are introduced at reverse primer 3 '-end the 2nd and the 3rd base
G.C.R)。
Table 2 is detected shows PCR primer used in the how main rod spore bacteria strain of resistance to fluopyram
Genomic DNA using how main rod spore bacterium to be measured is carried out as template using Cc1020F and Cc1020A/T/C/G.C.R
PCR is expanded, and 25 μ L amplification systems are：1 μ L template DNAs (30-50ng), every primer (10 μM, the concentration in L amplification systems
For 0.4 μM/L) 1 μ L, 12.5 μ 2 × Taq of L Mix (purchased from the rich Deco skill Development Co., Ltd advanced in years in Beijing), 9.5 μ L ddH2O。
Different annealing temperature grads PCR is carried out to each primer pair, it is determined that the high annealing temperature of specificity.Response procedures are：Pre-degeneration 94
℃5min；94 DEG C of 30s, 50-70 DEG C of 30s, 72 DEG C of 30s, 30 circulations；Extend 5min at last 72 DEG C.Amplified production is 2%
Electrophoresis detection in Ago-Gel.
As a result as shown in figure 3, S：Sensitive strain；R：Resistant strain；From top to bottom forward primer is followed successively by
Cc1020A.C.R, Cc1020T.C.R, Cc1020C.C.R, Cc1020G.C.R, forward primer are Cc1020F, primer pair
Cc1020F-Cc1020A/C.C.R can be from sensitive strain and the genome of resistant strain under 51.3-68.4 DEG C of annealing temperature
260bp fragment is amplified in DNA, it is impossible to well distinguish it.When annealing temperature is 61.4-68.4 DEG C, primer pair
Cc1020F-Cc1020T/G.C.R can amplify 260bp piece from resistant strain HG15050729-5 genomic DNA
Section.Wherein Cc1020F-Cc1020G.C.R primer pairs can also amplify faint band from sensitive strain at such a temperature, and
Cc1020F-Cc1020T.C.R can not amplify corresponding fragment from sensitive strain.
Therefore selection Cc1020F-Cc1020T.C.R primer pairs are used for detecting A1020G mutation position as AS-PCR primers
Point, annealing temperature is 61.4-68.4 DEG C.
By primer pair Cc1020F-Cc1020T.C.R antagonism bacterial strains HG15050729-5 amplified production 260bp piece
Duan Jinhang is sequenced, and as a result the nucleotides sequence of 260bp fragment is classified as in sequence 1 from 5 ' ends the 785th to 1044.
Therefore, detecting the kit of mutational site A1020G in how main rod spore bacterium includes Cc1020F and Cc1020T.C.R groups
Into primer pair；
Or mutational site A1020G kit includes containing Cc1020F and Cc1020T.C.R in the how main rod spore bacterium of detection
The PCR reagent of the primer pair of composition, the PCR reagent includes：Cc1020F, Cc1020T.C.R, 2 × Taq Mix and water, wherein
The concentration of Cc1020F, Cc1020T.C.R in reagent is respectively 0.4 μM/L and 0.4 μM/L.
2nd, the specific detection of primer pair
So that the genomic DNA for trying how main rod spore bacterium and other fungies and bacterium is template, using Cc1020F and
Cc1020T.C.R enters performing PCR amplification.
25 μ L amplification systems are：1 μ L template DNAs (30-50ng), every primer (0.4 μM/L) 1 μ L, 12.5 2 × Taq of μ L
Mix (purchased from the rich Deco skill Development Co., Ltd advanced in years in Beijing), 9.5 μ L ddH2O。
Response procedures are：94℃5min；94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 30s, 30 circulations；Extend 5min at 72 DEG C.
Amplified production electrophoresis detection in 2% Ago-Gel.
As a result as shown in figure 4, M：Marker (BM5000, the rich Deco skill Development Co., Ltd advanced in years in Beijing)；1, how main rod spore
Bacterium (Corynespora cassiicola) HG15050729-5 (resistance)；2, how main rod spore bacterium (C.cassiicola)
HG15050825-6 (resistance)；3, how main rod spore bacterium (C.cassiicola) HG14102524-4 (sensitivity)；4, phytophthora blight of pepper
(Phytophthora capsici)LJ12010805；5, eggplant disease reaping hook (Fusarium solani) HG13060501；6, coal is dirty
False tail spore (Pseudocercospora fuligena) FQ10013003；7, eggplant stemphylium botryosum (Stemphylium solani)
FQ14111912；8, how main rod spore bacterium (C.cassicola) HG15030802 (sensitivity)；9, Fusarium oxysporum (Fusarium
oxysporum)HG15060516；10, Rhizoctonia solani Kuhn (Rhizoctonia solani) DBC16070604；11, Botrytis cinerea
Bacterium (Botrytis cinerea) FQ13010701；12, sporulation (Alternaria solani) JGFQ15080702；
13, pseudomonas syringae cucumber pvs oryzae and oryzicola (Pseudomonas syringae pv.lachrymans) HG1501050702；
14, carrot soft rot Pectinatus Brazil's subspecies (Pectobacterium carotovorum subsp.brasiliensis)
HG0903060203；15, negative control "-"；Primer pair Cc1020F-Cc1020T.C.R is only capable of from carrying SdhB-V280 mutation
How main rod spore bacterial strain in amplify size be 260bp fragment.
3rd, the sensitivity technique of primer pair
(it is initial dense that NANO DROP 2000 determine it to the how main rod spore bacterium HG15050729-5 tried confession genomic DNA
Spend and be：9.9ng/ μ L) carry out 10 times of gradient dilutions (10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9), use
Cc1020F and Cc1020T.C.R enter performing PCR amplification.
25 μ L amplification systems are：1 μ L template DNAs (30-50ng), every primer (0.4 μM/L) 1 μ L, 12.5 2 × Taq of μ L
Mix (purchased from the rich Deco skill Development Co., Ltd advanced in years in Beijing), 9.5 μ L ddH2O。
Response procedures are：94 DEG C of 5min of pre-degeneration；94 DEG C of 30s, 68.4 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C downward
Stretch 5min.Amplified production electrophoresis detection in 2% Ago-Gel.
As a result as shown in figure 5, M：Marker (BM5000, the rich Deco skill Development Co., Ltd advanced in years in Beijing)；1,9.9ng/μL；
2,9.9×10-1ng/μL；3,9.9×10-2ng/μL；4,9.9×10-3ng/μL；5,9.9×10-4ng/μL；6,9.9×10- 5ng/μL；7,9.9×10-6ng/μL；8,9.9×10-7ng/μL；9,9.9×10-8ng/μL；10,9.9×10-9ng/μL；11,
Negative control "-", it can be seen that the detection sensitivity of the system is 9.9 × 10-2ng/μL。
4th, detect that the primer pair of mutational site A1020G in how main rod spore bacterium is detecting how main rod spore bacterium to fluopyram
Application in resistance
With how main rod spore bacterium HG15050755-3 to be measured, HG15041321-2, HG15041414-2, HG14102405-2,
HG14102524-4, HG15050729-5, HG15050731, HG15050737 and HG15050825-6 genomic DNA are mould
Plate, performing PCR amplification is entered using primer pair Cc1020F-Cc1020T.C.R.
PCR reaction systems are as follows：
25 μ L reaction systems include 1 μ L template DNAs (30-50ng), Cc1020F (0.4 μM/L) and Cc1020T.C.R
(0.4 μM/L) each 1 μ L, 12.5 μ 2 × Taq of L Mix (purchased from the rich Deco skill Development Co., Ltd advanced in years in Beijing), 9.5 μ L ddH2O。
Response procedures are：94 DEG C of 5min of pre-degeneration；94 DEG C of 30s, 68.4 DEG C of 30s, 72 DEG C of 30s, 30 circulations；Last 72 DEG C
Lower extension 5min.Amplified production electrophoresis detection in 2% Ago-Gel.
If obtaining 260bp fragment, succinate dehydrogenase B subunit gene SdhB nucleosides in the how main rod spore bacterium genome
The 1020th nucleotides of acid sequence is G, then the how main rod spore bacterium is or candidate is fluopyram resistant strain；
If not obtaining 260bp fragment, succinate dehydrogenase B subunit gene SdhB cores in the how main rod spore bacterium genome
The 1020th nucleotides of nucleotide sequence is not G, then the how main rod spore bacterium is not or candidate is not fluopyram resistant strain.
As a result as shown in fig. 6, M：Marker (BM5000, the rich Deco skill Development Co., Ltd advanced in years in Beijing)；1,
HG15050729-5；2,HG15050731；3,HG15050737；4,HG15050825-6；5,HG14102524-4；6,
HG14102405-2；7,HG15041321-2；8,HG15041414-2；9,HG15050755-3；10, negative control "-"；Primer
To Cc1020F-Cc1020T.C.R from how main rod spore bacteria strain HG15050729-5, HG15050731, HG15050737 and
HG15050825-6 amplifies about 260bp specific fragments, its be to fluopyram show resistance, this with above to fluorine pyrrole
Bacterium acid amides sensitivity Detection is consistent；
Primer pair Cc1020F-Cc1020T.C.R from how main rod spore bacteria strain HG15050755-3, HG15041321-2,
About 260bp specific fragments are not amplified in HG15041414-2, HG14102405-2 and HG14102524-4, it is to fluorine pyrrole
The performance of bacterium acid amides is sensitive, this with it is above consistent to fluopyram sensitivity Detection.
Show that this pair of primer can be used for whether the how main rod spore bacterium of specific detection generates resistance to fluopyram.
Sequence table
<110>Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science
<120>A kind of how main rod spore bacterium of Rapid identification is to the drug-fast method of fluopyram and special primer pair
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1106
<212> DNA
<213>More main rod spore bacterium (Corynespora cassiicola)
<400> 1
atggcttgca cacgcgcttt cgctcgtctg gctacgacgc ggaccggtac gtgaacccct 60
ctacgtgcac tacacaccca ttggagctct actgacagct ctccccgcag ccgtacggcc 120
cgctgccgtc ttcacgcgcg gttttgcctc cgtgaccgac acggccgccc gcgagcccgt 180
ctccaaggtg gccgagaaga tcgtgcccga cccggcccgc aaggtggttc ccgagagtca 240
gaccagcact gtcaaggacc cccagccgga caaggacgcg aagaccaaga ccttccacat 300
ctaccgctgg aacccggacg agcccacgtc gaagccaaag atgcagacgt acacgctgga 360
cctgaacaag accggcccca tgatgttgga tgcgctgatc cgcatcaaga acgagctcga 420
cccgaccttg acctttaggc gaagttgccg tgagggaatt tgtggaagct gcgcaatgaa 480
cattgacggc gtgaacacgc tggcctgtct ttgtacgtgg aaccgcagtc tccacgaatg 540
cggaattggg tggctaacga gtgcgcaaca ggccgcatcc ccaccgatac caccaaggaa 600
tcgcgcattt accctctccc tcacatgtac attgtcaagg acttggtccc cgacatgacg 660
ctcttctaca agcagtaccg ctccgtgaag ccctacctcc agcgcgacac tcctgctcct 720
gacgtaagaa caccgcacac ctcaccatag cctcgaactc gccactaacg attgctctct 780
agggccgtga ataccgccag tccaaggagg agcgcaagaa gctcgacggc ctgtacgagt 840
gcattctgtg cgcctgctgc tcaacgtcct gcccgtcgta ctggtggaac caggaggagt 900
acctcggccc tgccgtgctg ctccagtcgt accgctggat cgccgattcg cgtgacgaga 960
agacggcaca acgccaggat gccctcaaca actcgatgag catgtaccgc tgccacacgg 1020
ttctcaactg ctcgaggacg tgccccaagg gcttgaaccc ggctctggcc attgccgaaa 1080
tcaagaagag catggctttc acgtag 1106
<210> 2
<211> 307
<212> PRT
<213>More main rod spore bacterium (Corynespora cassiicola)
<400> 2
Met Ala Cys Thr Arg Ala Phe Ala Arg Leu Ala Thr Thr Arg Thr Ala
1 5 10 15
Val Arg Pro Ala Ala Val Phe Thr Arg Gly Phe Ala Ser Val Thr Asp
20 25 30
Thr Ala Ala Arg Glu Pro Val Ser Lys Val Ala Glu Lys Ile Val Pro
35 40 45
Asp Pro Ala Arg Lys Val Val Pro Glu Ser Gln Thr Ser Thr Val Lys
50 55 60
Asp Pro Gln Pro Asp Lys Asp Ala Lys Thr Lys Thr Phe His Ile Tyr
65 70 75 80
Arg Trp Asn Pro Asp Glu Pro Thr Ser Lys Pro Lys Met Gln Thr Tyr
85 90 95
Thr Leu Asp Leu Asn Lys Thr Gly Pro Met Met Leu Asp Ala Leu Ile
100 105 110
Arg Ile Lys Asn Glu Leu Asp Pro Thr Leu Thr Phe Arg Arg Ser Cys
115 120 125
Arg Glu Gly Ile Cys Gly Ser Cys Ala Met Asn Ile Asp Gly Val Asn
130 135 140
Thr Leu Ala Cys Leu Cys Arg Ile Pro Thr Asp Thr Thr Lys Glu Ser
145 150 155 160
Arg Ile Tyr Pro Leu Pro His Met Tyr Ile Val Lys Asp Leu Val Pro
165 170 175
Asp Met Thr Leu Phe Tyr Lys Gln Tyr Arg Ser Val Lys Pro Tyr Leu
180 185 190
Gln Arg Asp Thr Pro Ala Pro Asp Gly Arg Glu Tyr Arg Gln Ser Lys
195 200 205
Glu Glu Arg Lys Lys Leu Asp Gly Leu Tyr Glu Cys Ile Leu Cys Ala
210 215 220
Cys Cys Ser Thr Ser Cys Pro Ser Tyr Trp Trp Asn Gln Glu Glu Tyr
225 230 235 240
Leu Gly Pro Ala Val Leu Leu Gln Ser Tyr Arg Trp Ile Ala Asp Ser
245 250 255
Arg Asp Glu Lys Thr Ala Gln Arg Gln Asp Ala Leu Asn Asn Ser Met
260 265 270
Ser Met Tyr Arg Cys His Thr Val Leu Asn Cys Ser Arg Thr Cys Pro
275 280 285
Lys Gly Leu Asn Pro Ala Leu Ala Ile Ala Glu Ile Lys Lys Ser Met
290 295 300
Ala Phe Thr
305
<210> 3
<211> 1106
<212> DNA
<213>More main rod spore bacterium (Corynespora cassiicola)
<400> 3
atggcttgca cacgcgcttt cgctcgtctg gctacgacgc ggaccggtac gtgaacccct 60
ctacgtgcac tacacaccca ttggagctct actgacagct ctccccgcag ccgtacggcc 120
cgctgccgtc ttcacgcgcg gttttgcctc cgtgaccgac acggccgccc gcgagcccgt 180
ctccaaggtg gccgagaaga tcgtgcccga cccggcccgc aaggtggttc ccgagagtca 240
gaccagcact gtcaaggacc cccagccgga caaggacgcg aagaccaaga ccttccacat 300
ctaccgctgg aacccggacg agcccacgtc gaagccaaag atgcagacgt acacgctgga 360
cctgaacaag accggcccca tgatgttgga tgcgctgatc cgcatcaaga acgagctcga 420
cccgaccttg acctttaggc gaagttgccg tgagggaatt tgtggaagct gcgcaatgaa 480
cattgacggc gtgaacacgc tggcctgtct ttgtacgtgg aaccgcagtc tccacgaatg 540
cggaattggg tggctaacga gtgcgcaaca ggccgcatcc ccaccgatac caccaaggaa 600
tcgcgcattt accctctccc tcacatgtac attgtcaagg acttggtccc cgacatgacg 660
ctcttctaca agcagtaccg ctccgtgaag ccctacctcc agcgcgacac tcctgctcct 720
gacgtaagaa caccgcacac ctcaccatag cctcgaactc gccactaacg attgctctct 780
agggccgtga ataccgccag tccaaggagg agcgcaagaa gctcgacggc ctgtacgagt 840
gcattctgtg cgcctgctgc tcaacgtcct gcccgtcgta ctggtggaac caggaggagt 900
acctcggccc tgccgtgctg ctccagtcgt accgctggat cgccgattcg cgtgacgaga 960
agacggcaca acgccaggat gccctcaaca actcgatgag catgtaccgc tgccacacga 1020
ttctcaactg ctcgaggacg tgccccaagg gcttgaaccc ggctctggcc attgccgaaa 1080
tcaagaagag catggctttc acgtag 1106
<210> 4
<211> 307
<212> PRT
<213>More main rod spore bacterium (Corynespora cassiicola)
<400> 4
Met Ala Cys Thr Arg Ala Phe Ala Arg Leu Ala Thr Thr Arg Thr Ala
1 5 10 15
Val Arg Pro Ala Ala Val Phe Thr Arg Gly Phe Ala Ser Val Thr Asp
20 25 30
Thr Ala Ala Arg Glu Pro Val Ser Lys Val Ala Glu Lys Ile Val Pro
35 40 45
Asp Pro Ala Arg Lys Val Val Pro Glu Ser Gln Thr Ser Thr Val Lys
50 55 60
Asp Pro Gln Pro Asp Lys Asp Ala Lys Thr Lys Thr Phe His Ile Tyr
65 70 75 80
Arg Trp Asn Pro Asp Glu Pro Thr Ser Lys Pro Lys Met Gln Thr Tyr
85 90 95
Thr Leu Asp Leu Asn Lys Thr Gly Pro Met Met Leu Asp Ala Leu Ile
100 105 110
Arg Ile Lys Asn Glu Leu Asp Pro Thr Leu Thr Phe Arg Arg Ser Cys
115 120 125
Arg Glu Gly Ile Cys Gly Ser Cys Ala Met Asn Ile Asp Gly Val Asn
130 135 140
Thr Leu Ala Cys Leu Cys Arg Ile Pro Thr Asp Thr Thr Lys Glu Ser
145 150 155 160
Arg Ile Tyr Pro Leu Pro His Met Tyr Ile Val Lys Asp Leu Val Pro
165 170 175
Asp Met Thr Leu Phe Tyr Lys Gln Tyr Arg Ser Val Lys Pro Tyr Leu
180 185 190
Gln Arg Asp Thr Pro Ala Pro Asp Gly Arg Glu Tyr Arg Gln Ser Lys
195 200 205
Glu Glu Arg Lys Lys Leu Asp Gly Leu Tyr Glu Cys Ile Leu Cys Ala
210 215 220
Cys Cys Ser Thr Ser Cys Pro Ser Tyr Trp Trp Asn Gln Glu Glu Tyr
225 230 235 240
Leu Gly Pro Ala Val Leu Leu Gln Ser Tyr Arg Trp Ile Ala Asp Ser
245 250 255
Arg Asp Glu Lys Thr Ala Gln Arg Gln Asp Ala Leu Asn Asn Ser Met
260 265 270
Ser Met Tyr Arg Cys His Thr Ile Leu Asn Cys Ser Arg Thr Cys Pro
275 280 285
Lys Gly Leu Asn Pro Ala Leu Ala Ile Ala Glu Ile Lys Lys Ser Met
290 295 300
Ala Phe Thr
305
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
ccgtgaatac cgccagtcca a 21
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400>6
ggcacgtcct cgagcagttg agtcc 25
Claims (10)
1. the material of the succinate dehydrogenase B subunit coding gene A1020G sites base in the how main rod spore bacterium genome of detection exists
The how main rod spore bacterium of detection is to the application in fluopyram resistance；
The A1020G sites are the 1020th in the succinate dehydrogenase B subunit coding genes；
The base in the A1020G sites is A or G.
2. the material of the amino acids residue of succinate dehydrogenase B subunits the 280th in the how main rod spore bacterium genome of detection is in detection
More main rod spore bacterium is to the application in fluopyram resistance；
The 280th amino acids residue is the succinate dehydrogenase B yldeneaminos acid sequence the 280th；
The 280th amino acids residue is Ile or Val.
3. a kind of detection or the how main rod spore bacterium of auxiliary detection comprise the following steps to the method for fluopyram resistance：Detection is treated
The succinate dehydrogenase B subunit coding gene A1020G sites base in genome in how main rod spore bacterium is surveyed, it is described A1020G
The base of point is A or G；If the base in the site is A, the how main rod spore bacterium is or candidate is sensitive to fluopyram；If
The amino acid residue in the site is G, then the how main rod spore bacterium is or candidate is resistant to fluopyram.
4. a kind of detection or the how main rod spore bacterium of auxiliary detection comprise the following steps to the method for fluopyram resistance：Detection is treated
The amino acid for surveying the 280th, the albumen that the succinate dehydrogenase B subunit coding genes in how main rod spore bacterium in genome are encoded is residual
Base, the 280th amino acids residue is Ile or Val；If the amino acid residue in the site is Ile, the how main rod spore bacterium is
Or candidate is sensitive to fluopyram；If the amino acid residue in the site is Val, the how main rod spore bacterium is or candidate is pair
Fluopyram is resistant.
5. a kind of detection or the how main rod spore bacterium of auxiliary detection comprise the following steps to the method for fluopyram resistance：Detection is treated
The succinate dehydrogenase B subunit coding gene A1020G sites base in genome in how main rod spore bacterium is surveyed, it is described A1020G
The base of point is A or G；The base in the site is higher than the site for G how main rod spore bacterium to be measured to the resistance of fluopyram
Base is A how main rod spore bacterium to be measured.
6. a kind of detection or the how main rod spore bacterium of auxiliary detection comprise the following steps to the method for fluopyram resistance：Detection is treated
Survey the amino acid of the 280th of the albumen that the succinate dehydrogenase B subunit coding genes in how main rod spore bacterium in genome are encoded
Residue, the 280th amino acids residue is Ile or Val；The amino acid residue in the site is Val how main rod spore bacterium to be measured
Resistance to fluopyram is higher than to be measured how main rod spore bacterium of the amino acid residue in the site for isoleucine.
7. according to any described method in claim 3-6, it is characterised in that：
The albumen of succinate dehydrogenase B subunit coding genes coding in the detection how main rod spore bacterium to be measured in genome the
The amino acid residue of 280 is by detecting the succinate dehydrogenase B subunit coding genes in how main rod spore bacterium to be measured in genome
A1020G sites base is realized；
Succinate dehydrogenase B subunit coding gene A1020G sites base in the detection how main rod spore bacterium to be measured in genome
1) or 2) realized by following method：
1) it is sequenced；
2) with the succinate dehydrogenase B subunit coding gene A1020G sites base in the how main rod spore bacterium genome of detection
Material expands the how main rod spore bacterium to be measured, amplified production is detected, if the amplified production obtains specific fragment, the detection
Succinate dehydrogenase B subunit coding gene A1020G sites base in how main rod spore bacterium to be measured in genome is G, if amplification production
Thing does not obtain specific fragment, then the succinate dehydrogenase B subunits coding base in the detection how main rod spore bacterium to be measured in genome
Because A1020G sites base is A；
The material of succinate dehydrogenase B subunit coding gene A1020G sites base in the how main rod spore bacterium genome of detection
Including following A or B：
A the primer pair in the A1020G sites) is expanded；
B) PCR reagent or kit containing the primer pair.
8. method according to claim 7, it is characterised in that：
The primer pair in the amplification A1020G sites is made up of the nucleotides shown in the nucleotides shown in sequence 5 and sequence 6；
Or the specific fragment size is 250-270bp；
Or the annealing temperature of the amplification is 61.4-68.4 DEG C.
9. applied described in claim 1 or 2 or any methods describeds of claim 3-9 in the how main rod spore bacterium of the detection to fluorine
The material of pyrrole bacterium acid amides resistance；
Or a kind of succinate dehydrogenase B subunit coding gene A1020G sites alkali detected in how main rod spore bacterium to be measured in genome
The method of base, comprises the following steps：With the succinate dehydrogenase B subunit coding genes in the how main rod spore bacterium genome of detection
The material of A1020G sites base expands the how main rod spore bacterium to be measured, amplified production is detected, if the amplified production obtains spy
Heteroleptic, then succinate dehydrogenase B subunit coding genes A1020G in the detection how main rod spore bacterium to be measured in genome
Point base is G, if amplified production does not obtain the butanedioic acid in genome in specific fragment, the detection how main rod spore bacterium to be measured
Dehydrogenase B subunit coding gene A1020G sites base is A；
The material of succinate dehydrogenase B subunit coding gene A1020G sites base in the how main rod spore bacterium genome of detection
Including following A or B：
A the primer pair in the A1020G sites) is expanded；
B) PCR reagent or kit containing the primer pair.
10. method according to claim 9, it is characterised in that：
The primer pair in the amplification A1020G sites is made up of the nucleotides shown in the nucleotides shown in sequence 5 and sequence 6；
Or the specific fragment size is 250-270bp；
Or the annealing temperature of the amplification is 61.4-68.4 DEG C.
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Cited By (6)
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| CN107988354A (en) * | 2017-12-13 | 2018-05-04 | 广州迈景基因医学科技有限公司 | Primer, kit and the method for NUDT15 Genotypings |
| CN110857452A (en) * | 2018-08-23 | 2020-03-03 | 中国农业科学院蔬菜花卉研究所 | Method for detecting content of SdhB-H278R point mutation and special reagent set thereof |
| CN111996279A (en) * | 2020-08-31 | 2020-11-27 | 中国农业科学院蔬菜花卉研究所 | Method for detecting succinate dehydrogenase D subunit point mutation type of corynebacterium polygamum and primer composition used in method |
| CN113481216A (en) * | 2021-08-06 | 2021-10-08 | 中国农业科学院蔬菜花卉研究所 | Polysorium CcTLS1 protein, coding gene and application thereof |
| CN115786560A (en) * | 2022-08-12 | 2023-03-14 | 上海市农业科学院 | Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of botrytis cinerea |
| CN116397047A (en) * | 2023-05-10 | 2023-07-07 | 三亚中国农业科学院国家南繁研究院 | Primer combination for detecting B-I280V mutation of corynespora polymorpha |
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| CN107988354A (en) * | 2017-12-13 | 2018-05-04 | 广州迈景基因医学科技有限公司 | Primer, kit and the method for NUDT15 Genotypings |
| CN107988354B (en) * | 2017-12-13 | 2021-05-14 | 广州迈景基因医学科技有限公司 | Primer, kit and method for NUDT15 genotyping |
| CN110857452A (en) * | 2018-08-23 | 2020-03-03 | 中国农业科学院蔬菜花卉研究所 | Method for detecting content of SdhB-H278R point mutation and special reagent set thereof |
| CN111996279A (en) * | 2020-08-31 | 2020-11-27 | 中国农业科学院蔬菜花卉研究所 | Method for detecting succinate dehydrogenase D subunit point mutation type of corynebacterium polygamum and primer composition used in method |
| CN113481216A (en) * | 2021-08-06 | 2021-10-08 | 中国农业科学院蔬菜花卉研究所 | Polysorium CcTLS1 protein, coding gene and application thereof |
| CN113481216B (en) * | 2021-08-06 | 2023-08-15 | 中国农业科学院蔬菜花卉研究所 | Multi-main corynespora CcTLS1 protein and coding gene and application thereof |
| CN115786560A (en) * | 2022-08-12 | 2023-03-14 | 上海市农业科学院 | Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of botrytis cinerea |
| CN115786560B (en) * | 2022-08-12 | 2024-03-22 | 上海市农业科学院 | Primer group, kit and method for detecting B subunit point mutation type of succinate dehydrogenase of Botrytis cinerea |
| CN116397047A (en) * | 2023-05-10 | 2023-07-07 | 三亚中国农业科学院国家南繁研究院 | Primer combination for detecting B-I280V mutation of corynespora polymorpha |
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