CN104404158B - A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament - Google Patents
A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament Download PDFInfo
- Publication number
- CN104404158B CN104404158B CN201410744410.7A CN201410744410A CN104404158B CN 104404158 B CN104404158 B CN 104404158B CN 201410744410 A CN201410744410 A CN 201410744410A CN 104404158 B CN104404158 B CN 104404158B
- Authority
- CN
- China
- Prior art keywords
- citrus
- pathogenic bacteria
- yellow
- chemical agent
- psylla
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the triage techniques of chemical prevention medicament in agriculture field, specifically disclose the rapid screening method of a kind of citrus yellow shoot disease prevention and control medicament.The preliminary assessment material of described citrus yellow shoot disease prevention and control medicament rapid screening method is the citrus psylla that carries disease germs, and uses yellow twig pathogenic bacteria growth and decline degree in the citrus psylla body that carries disease germs described in RT qPCR real-time dynamic monitoring to evaluate drug effect.The present invention creatively utilizes the citrus psylla screening material as anti-yellow twig pathogenic bacteria chemical agent that carries disease germs, at short notice potentially large number of citrus yellow shoot disease prevention and control medicament can be carried out rapid screening and evaluation, solve current anti-yellow twig pathogenic bacteria chemical agent screening excessive cycle, lack the deficiencies such as a set of accurate and effective appraisement system.
Description
Technical field
The present invention relates to the triage techniques of chemical prevention medicament in agriculture field, more specifically, relate to a kind of mandarin orange HUANGLONG
The rapid screening method of sick prevention and control medicament.
Background technology
Citrus yellow shoot disease occurs with a long history in China, endangers the most serious.Have made be Reinking in
Within 1919, find citrus yellow shoot disease first in Chao-Shan Area, China Guangdong.To 20th century the mid-80, citrus yellow shoot disease exists
The mandarin orange producing region extensive widespread in China Guangdong, Guangxi, Fujian, Hainan and Taiwan, and in Zhejiang, Guizhou, the ground part Citrus chachiensis Hort. such as Hunan
Fructus Citri tangerinae producing region occurs in succession.China has 19 provinces of citrus culture, existing 11 of district to be endangered, and injured area accounts for mandarin orange and always cultivates
More than the 80% of area, about 85% (model state one-tenth etc., 2009) of total output.But, in view of yellow twig pathogenic bacteria
(' CandidatusLiberibacter asiaticus ' is called for short Las) the difficult characteristic cultivated, relative chemical agent
Screening operation is the most still made slow progress.
The anti-yellow twig pathogenic bacteria chemical agent screening experiment carried out for citrus yellow shoot disease chemical prevention.At present, in state
Inside and outside be in progress.But, screening technique in early days is typically based on the symptom variation for morbidity plant itself, often
Lack a set of accurate and effective appraisement system, and it is longer (it is generally required to 1-3 is the most long to there is also the screening cycle
Continuously monitoring), easily it is affected by the external environment and waits deficiency.As: trunk injection apparatus, phloem method of topical application, foliar spray method and soil
Pouring method etc..Until in recent years, in view of the harm situation day of citrus yellow shoot disease is the severeest, and present stage yellow twig pathogenic bacteria is the most not
Can successfully carry out separation and Culture, just make more scholar efforts be made so that with other disease sources of citrus yellow shoot disease and prevent and treat as it
The screening and assessment system of medicament.Such as Muqing Zhang(2010,2011) etc. the Herba Catharanthi Rosei branch having infected yellow twig is utilized to exist
Survival rate after chemicals treatment evaluates drug effect, constructs yellow twig chemical prevention medicament primary dcreening operation system based on Herba Catharanthi Rosei.Should
Though primary dcreening operation system has substantially reduced screening time, but still needs at least 60-90 d and could obtain preliminary the selection result.For Zong
It, current anti-yellow twig pathogenic bacteria chemical agent screening excessive cycle, and lack a set of accurate and effective appraisement system.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that the rapid screening method of a kind of citrus yellow shoot disease prevention and control medicament.
The present invention creatively utilizes the citrus psylla screening material as anti-yellow twig pathogenic bacteria chemical agent that carries disease germs, can be in the short time
In citrus yellow shoot disease prevention and control medicament is carried out rapid screening and evaluation, solve current anti-yellow twig pathogenic bacteria chemical agent screening week
Phase is long, lacks the deficiencies such as a set of accurate and effective appraisement system.
The purpose of the present invention is achieved by the following technical programs.
A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament, the preliminary assessment of described citrus yellow shoot disease prevention and control medicament
Material is the citrus psylla that carries disease germs, and uses yellow twig pathogenic bacteria described in RT-qPCR real-time dynamic monitoring in the citrus psylla body that carries disease germs
Growth and decline degree evaluate drug effect.
Prior art constructs yellow twig chemical prevention medicament primary dcreening operation system based on Herba Catharanthi Rosei, utilizes PCR to detect skill
Art, with containing of the Candidatus liberibacter of the regeneration biological amount that survives of after Chemical treatment 2 ~ 6 month Herba Catharanthi Roseis and newborn long branch
Amount is index, filters out the medicine of anti-Candidatus liberibacter, and screening time is long, it is understood that there may be screen inaccurate situation.And this
The bright citrus psylla screening material as anti-yellow twig pathogenic bacteria chemical agent that carries disease germs that creatively utilizes, and use RT-qPCR real
Time dynamic monitoring described in yellow twig pathogenic bacteria growth and decline degree in the citrus psylla body that carries disease germs, it is only necessary within 5 days, can filter out effectively anti-
The chemical agent of citrus yellow shoot disease pathogenic bacteria, screening time is short, accuracy is high, it is provided that the most anti-yellowing dragon of one
Sick pathogenic bacteria chemical agent screening technique, solves current anti-yellow twig pathogenic bacteria chemical agent screening excessive cycle, lacks an alignment
Really, the deficiency such as effectively evaluating system.
Preferably, described method comprises the steps:
S1. process is fed: after the citrus psylla imago feeding appropriate chemical agent solution that carries disease germs, then the mandarin orange leaf by health
Sheet is raised；
S2. evaluating drug effect: extract the citrus psylla STb gene that carries disease germs after feeding process, more real-time by RT-qPCR technology
Dynamic monitoring yellow twig pathogenic bacteria growth and decline degree in wood louse body carrys out preliminary assessment drug effect.
Preferably, the concentration of described chemical agent solution is 1000 mg/L.
Preferably, the process time of described chemical agent is 24h.
Preferably, described wood louse adult is used further to S1 step after Nature enemy 12h.
Preferably, in described S2 step, the detection primer of RT-qPCR real-time dynamic monitoring is HLBasf/ HLBasr,
Its nucleotide sequence is respectively as shown in SEQ ID NO:1 ~ 2；Probe is HLBp, and its nucleotide sequence is as shown in SEQ ID NO:3；
Described detection reaction system is 2 × TaqMan PCR Master Mix 10 μ L, and the upstream and downstream of 10 μm ol/L is drawn
Thing and probe each 0.4 μ L, DNA profiling 1 μ L, then add sterilizing ddH2O is to cumulative volume 20 μ L, and reaction condition is 95 DEG C of denaturations
After 30 s；95 DEG C of annealing 5 s, 60 DEG C extend 30 s, totally 40 circulations；Synchronize in real time in annealing and the extension stage of each circulation
Gather fluorescence, according to standard curve, converse the content of yellow twig cause of disease in testing sample.
The present invention is found through experiments, after 5d processes, filter out quadracycline in tetracycline antibiotics and
Tetramycin hydrochloride, gentamycin sulfate and amikacin sulfate in aminoglycoside antibiotics are that effective anti-citrus yellow shoot disease is sick
The chemical agent of bacterium.
The present invention also provides for the application of the rapid screening method of a kind of Anti-bacterium chemical agent, the evaluation material of described method
For the disease source insecticide of its pathogenic bacteria, and use the growth and decline journey in the insect bodies that carries disease germs of the pathogenic bacteria described in RT-qPCR real-time dynamic monitoring
Degree evaluates drug effect, and described method is applied to the difficult sieve cultivating pathogenic bacteria chemical prevention medicament that sucking pest is propagated
Choosing.
Compared with prior art, the beneficial effects of the present invention is:
(1) present invention creatively utilizes the citrus psylla screening material as anti-yellow twig pathogenic bacteria chemical agent that carries disease germs,
At short notice citrus yellow shoot disease prevention and control medicament can be carried out rapid screening and evaluation, solve current anti-yellow twig pathogenic bacteriaization
Learn Chemicals excessive cycle, lack the deficiencies such as a set of accurate and effective appraisement system.
(2) present invention uses the citrus psylla preliminary assessment material as anti-yellow twig pathogenic bacteria chemical agent that carries disease germs, and passes through
Real-Time Fluorescent Quantitative PCR Technique (RT-qPCR) real-time dynamic monitoring yellow twig pathogenic bacteria evaluates medicine in its internal growth and decline degree
Effect, it is only necessary to the chemical agent of effective anti-citrus yellow shoot disease pathogenic bacteria can be filtered out for 5 days, it is provided that one is the most fast and effectively
Anti-yellow twig pathogenic bacteria chemical agent screening technique, the efficient citrus yellow shoot disease prevention and control medicament that screening obtains, can not only be used for producing
Citrus yellow shoot disease chemical prevention medicament directly utilized, can be also the research preventing and treating citrus yellow shoot disease pharmaceutical preparations and open
Send out, and the research work such as citrus yellow shoot disease pathogenic bacteria and host's interaction mechanism provides theoretical reference.
Accompanying drawing explanation
Fig. 1 is that different time processes the impact of yellow twig pathogenic bacteria concentration in citrus psylla body.
Fig. 2 is that tetracycline antibiotics is to the Antibacterial Activity result of yellow twig pathogenic bacteria in citrus psylla body.
Fig. 3 is that aminoglycoside antibiotics is to the Antibacterial Activity result of yellow twig pathogenic bacteria in citrus psylla body.
Fig. 4 is that beta-lactam antibiotic is to the Antibacterial Activity result of yellow twig pathogenic bacteria in citrus psylla body.
Fig. 5 is that Comprecin and other antimicrobial drugs are to the Antibacterial Activity of yellow twig pathogenic bacteria in citrus psylla body
Result.
Fig. 6 is that quadracycline and tetramycin hydrochloride are to the removal effect of yellow twig pathogenic bacteria in citrus psylla body.
Caption (Fig. 1-3): CK represents blank group；* represent and there is significant difference (P ＜ 0.05) through t inspection；* represents
Extremely significant difference (P ＜ 0.01) is there is through t inspection.
Detailed description of the invention
Below in conjunction with Figure of description and specific embodiment, the present invention is described in further details, but embodiment is the most right
The present invention limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus are that the art is normal
Rule reagent, method and apparatus.
The specific embodiment of the invention is mainly used in the rapid screening of anti-yellow twig pathogenic bacteria chemical agent, and suitable
Changing for examination insecticide, chemical agent kind and feeding device, the method for the invention can also be applied to other pierce-suck type mouths
The difficult screening cultivating pathogenic bacteria chemical prevention medicament that device insect is propagated.
The rapid screening process of embodiment 1 anti-yellow twig pathogenic bacteria chemical agent
Take the citrus psylla that carries disease germs after 15 hunger 12 h, with feeding for examination chemical agent solution of 3 mL 1000 mg/L
24h, then raise by healthy citrus leaves.Rearing conditions: temperature is 26-28 DEG C, relative humidity is 60%-75%, photoperiod L:D=
14: 10 h.In experimentation, check once the moisturizing situation of healthy citrus leaves every 24 h, and be simultaneously introduced appropriate phase
Answering chemical agent solution to carry out moisturizing process, causing carrying disease germs citrus psylla to prevent healthy citrus leaves excessive dehydration cannot be just
Often take food.With the distilled water without any chemical agent as blank, process every time and be respectively provided with 3 repetitions.Chemical agent
Feed after process completes, use OMEGA®E.Z.N.A. Insect DNA Kit test kit (D0926-01) that company buys
Extraction is carried disease germs citrus psylla STb gene, and recycling RT-qPCR technology real-time dynamic monitoring yellow twig pathogenic bacteria is in its internal growth and decline journey
Degree carrys out preliminary assessment drug effect.
The RT-qPCR of Las detects primer the HLBasf/HLBasr(5 '-TCG reported with reference to (2006) such as Li
AGCGCGTATGCAATACG-3 '/5 '-GCGTTATCCCGTAGAAAAAGGTAG-3 '), its nucleotide sequence is respectively such as SEQ
Shown in ID NO:1, SEQ ID NO:2；Probe HLBp(5 '-FAM-AGACGGGTGAGTAACGCG-BHQ-3 '), its nucleotides sequence
Row probe and primer as shown in SEQ ID NO:3 are synthesized by Dalian treasured biological engineering company limited.RT-qPCR reaction system
For: 2 × TaqMan PCR Master Mix 10 μ L, the upstream and downstream primer of 10 μm ol/L and each 0.4 μ L of probe, DNA profiling
1 μ L, then add sterilizing ddH2O to cumulative volume 20 μ L.Often wheel reaction is respectively provided with the standard substance (A4 plasmid) of 7 gradients as outer ginseng
Draw standard curve, ddH2O is blank, and healthy mandarin orange DNA sample is negative control, and experiment is repeated 3 times above.Reaction
Carrying out in CFX96 Touch quantitative fluorescent PCR detecting system, reaction condition is: after 95 DEG C of denaturation 30 s, 40
Circulation (95 DEG C of annealing 5 s；60 DEG C, extend 30 s), in the annealing of each circulation with extend the stage and synchronize Real-time Collection fluorescence.Journey
Sequence is arranged and result reads and all uses Bio-Rad CFX Manger 2.1 software.According to the standard curve of ginseng outside A4 plasmid, change
Calculate the content of yellow twig cause of disease in testing sample.
Embodiment 2 different time processes the impact of yellow twig pathogenic bacteria concentration in citrus psylla body
The different disposal time of research quadracycline is to the fungistatic effect of yellow twig pathogenic bacteria in the citrus psylla body that carries disease germs
Impact.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing quadracycline, then divide by healthy citrus leaves
Not raising 1,3,5,7 and 9 d, experimental result is shown in Fig. 1, and in citrus psylla body, the suppression ratio of yellow twig pathogenic bacteria concentration is respectively as follows:
14.19%, 25.54%, 59.17%, 68.88% and 89.10%；And the concentration of yellow twig pathogenic bacteria in blank group citrus psylla body
Rise-1.55%, 25.51%, 29.21%, 39.89% and 37.86% the most respectively.And after 3 d process, process group citrus psylla
Significant difference (P < 0.05) is there is between suppression ratio and the blank group of internal yellow twig pathogenic bacteria concentration；At 5,7 and 9 d
After reason, in process group citrus psylla body, between suppression ratio and the blank group of yellow twig pathogenic bacteria concentration, there is pole significant difference
(P < 0.01).Meanwhile, in discovery citrus psylla body, the change of yellow twig pathogenic bacteria concentration is the most obvious at 3 d and 5 d process.Cause
This, preliminary selected 5 d are its optimization process natural law.Thereafter, other all chemical agent solution are all by 5 d process.
Embodiment 3 tetracycline antibiotics is on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria in the citrus psylla body that carries disease germs
Research tetracycline antibiotics is on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria in citrus psylla body, and experimental result is shown in
Fig. 2.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing hydrochloric acid Klinomycin, then raise by healthy citrus leaves
Supporting 5 d, the suppression ratio of its internal yellow twig pathogenic bacteria concentration is 5.58%, there was no significant difference before processing；When the citrus psylla that carries disease germs
Take food after containing the chemical agent solution of quadracycline or tetramycin hydrochloride respectively, the suppression of its internal yellow twig pathogenic bacteria concentration
Rate is respectively 59.17% and 49.70%, and there is pole significant difference (P < 0.01) before processing.But, when the citrus psylla that carries disease germs
After taking food the chemical agent solution containing doxycycline hydrochloride, its internal yellow twig pathogenic bacteria concentration but rises 34.90%.This table
Bright, quadracycline and tetramycin hydrochloride in tetracycline antibiotics all can effectively suppress yellow twig pathogenic bacteria at citrus psylla body
Internal breeding, and the yellow twig pathogenic bacteria concentration carried disease germs in citrus psylla body can be significantly reduced in pole；Though hydrochloric acid Klinomycin also can be one
Determining to suppress yellow twig pathogenic bacteria at citrus psylla proliferation in vivo in degree, but it is to yellow twig pathogenic bacteria concentration in the citrus psylla body that carries disease germs
Reduction effect inconspicuous；And doxycycline hydrochloride can not effectively suppress yellow twig pathogenic bacteria propagation in citrus psylla body.
Result shows, quadracycline and tetramycin hydrochloride are filter out from this screening tetracycline antibiotics used the most anti-
Citrus yellow shoot disease pathogenic bacteria chemical agent.
Embodiment 4 aminoglycoside antibiotics is on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria in citrus psylla body
Research aminoglycoside antibiotics is on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria, experimental result in citrus psylla body
See Fig. 3.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing streptomycin sulfate, then raise by healthy citrus leaves
Supporting 5 d, the suppression ratio of its internal yellow twig pathogenic bacteria concentration is 6.97%；And it is molten to take food the chemical agent containing kanamycin sulfate
After liquid, the concentration of its internal yellow twig pathogenic bacteria then rises 6.22%, but all there was no significant difference with before processing.When the mandarin orange that carries disease germs
After wood louse takes food the chemical agent solution containing gentamycin sulfate or amikacin sulfate respectively, its internal yellow twig pathogenic bacteria is dense
The suppression ratio of degree is respectively 41.21% and 42.69%, and there is pole significant difference (P < 0.01) before processing.This shows, amino
Gentamycin sulfate and amikacin sulfate in sugar tobramycin antibiotic all can effectively suppress yellow twig pathogenic bacteria at citrus psylla body
Internal breeding, and the yellow twig pathogenic bacteria concentration carried disease germs in citrus psylla body can be significantly reduced in pole；And though streptomycin sulfate also can be one
Determining to suppress yellow twig pathogenic bacteria at citrus psylla proliferation in vivo in degree, but it is to yellow twig pathogenic bacteria concentration in the citrus psylla body that carries disease germs
Reduction effect inconspicuous；And kanamycin sulfate can not effectively suppress yellow twig pathogenic bacteria propagation in citrus psylla body.
Result shows, gentamycin sulfate and amikacin sulfate are to filter out from this screening aminoglycoside antibiotics used
Effective anti-citrus yellow shoot disease pathogenic bacteria chemical agent.
Embodiment 5 beta-lactam antibiotic is on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria in citrus psylla body
Research beta-lactam antibiotic is on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria, experimental result in citrus psylla body
See Fig. 4.After the citrus psylla that carries disease germs takes food and contains chemical agent solution 24 h of ampicillin or ceftazidime respectively, then with strong
Health citrus leaves raises 5 d respectively, and the suppression ratio of its internal yellow twig pathogenic bacteria concentration is respectively 7.40% and 21.71%, but all with
Without significance sex differernce before processing；When after the chemical agent solution that the citrus psylla that carries disease germs takes food containing benzylpenicillin, its internal HUANGLONG
Sick pathogenic bacteria concentration but rises 39.60%.This shows, though ampicillin in beta-lactam antibiotic and ceftazidime can be
Suppression yellow twig pathogenic bacteria is at citrus psylla proliferation in vivo to a certain extent, but to yellow twig pathogenic bacteria concentration in the citrus psylla body that carries disease germs
Reduction effect inconspicuous；And benzylpenicillin can not effectively suppress yellow twig pathogenic bacteria propagation in citrus psylla body.Result table
Bright, this screening not yet, from the beta-lactam antibiotic used by this screening, filters out effective anti-citrus yellow shoot disease sick
Bacterium chemical agent.
Embodiment 6 Comprecins etc. are on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria in citrus psylla body
Research Comprecins etc. are on the impact of the bacteriostatic activity of yellow twig pathogenic bacteria in citrus psylla body, experimental result
See Fig. 5.Ciprofloxacin, pefloxacin, soluble chloride mycin or benziothiazolinone is contained respectively when the citrus psylla that carries disease germs takes food
After chemical agent solution 24 h, then raising 5 d respectively by healthy citrus leaves, its internal yellow twig pathogenic bacteria concentration rises respectively
12.37%, 30.84%, 13.02% and 26.13%.This shows, the ciprofloxacin in Comprecin, pefloxacin,
Soluble chloride mycin in chloromycetin series antibiotics and benziothiazolinone etc. all can not effectively suppress yellow twig pathogenic bacteria at citrus psylla
Internal propagation.Result shows, this screening is not yet from the Comprecin used by this screening and chloromycetin antibiosis
In element, filter out effective anti-citrus yellow shoot disease pathogenic bacteria chemical agent.
Embodiment 7 quadracycline and tetramycin hydrochloride are to the removal effect of yellow twig pathogenic bacteria in citrus psylla body
Research quadracycline and tetramycin hydrochloride are to the removal effect of yellow twig pathogenic bacteria, experimental result in citrus psylla body
See Fig. 6.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing quadracycline, then divide by healthy citrus leaves
Do not raise 15 d(to survive 8) and 20 d(survival 3), the suppression ratio of its internal yellow twig pathogenic bacteria concentration is respectively 98.36% He
100.00%；And after the citrus psylla that carries disease germs takes food chemical agent solution 24 h containing tetramycin hydrochloride, then by healthy citrus leaves
Raise 15 d(respectively to survive 11) and 20 d(survival 5), the suppression ratio of its internal yellow twig pathogenic bacteria concentration is respectively
95.87% and 99.29%.Experimental result tentatively shows, carry disease germs the yellow twig pathogenic bacteria in citrus psylla body, can be by taking for a long time
Food removes containing quadracycline or the chemical agent solution of tetramycin hydrochloride.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>rapid screening method of a kind of citrus yellow shoot disease prevention and control medicament
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>primer HLBasf
<400> 1
tcgagcgcgt atgcaatacg 20
<210> 2
<211> 24
<212> DNA
<213>primer HLBasr
<400> 2
gcgttatccc gtagaaaaag gtag 24
<210> 3
<211> 18
<212> DNA
<213>probe HLBp
<400> 3
agacgggtga gtaacgcg 18
Claims (5)
1. the rapid screening method of a citrus yellow shoot disease prevention and control medicament, it is characterised in that described citrus yellow shoot disease prevention and control medicament
The preliminary assessment material of rapid screening method is the citrus psylla that carries disease germs, and uses yellow twig described in RT-qPCR real-time dynamic monitoring
Pathogenic bacteria growth and decline degree in the citrus psylla body that carries disease germs is to evaluate drug effect；Comprise the steps:
S1. feed process: after the citrus psylla imago feeding appropriate chemical agent solution that carries disease germs, then raise by healthy citrus leaves
Support；
S2. evaluating drug effect: extract the citrus psylla STb gene that carries disease germs after feeding process, then by RT-qPCR technology Real-time and Dynamic
Monitoring yellow twig pathogenic bacteria growth and decline degree in wood louse body carrys out preliminary assessment drug effect.
Method the most according to claim 1, it is characterised in that the concentration of described chemical agent solution is 1000 mg/L.
Method the most according to claim 2, it is characterised in that the process time of described chemical agent is 24h.
Method the most according to claim 3, it is characterised in that described wood louse adult is used further to after Nature enemy 12h
S1 step.
Method the most according to claim 4, it is characterised in that the inspection of RT-qPCR real-time dynamic monitoring in described S2 step
Survey primer is HLBasf/HLBasr, and its nucleotide sequence is respectively as shown in SEQ ID NO:1 ~ 2；Probe is HLBp, its nucleotide
Sequence is as shown in SEQ ID NO:3；
Described detection reaction system is 2 × TaqMan PCR Master Mix 10 μ L, the upstream and downstream primer of 10 μm ol/L and
Probe each 0.4 μ L, DNA profiling 1 μ L, then add sterilizing ddH2O is to cumulative volume 20 μ L, and reaction condition is 95 DEG C of denaturation 30 s
After；95 DEG C of annealing 5 s, 60 DEG C extend 30 s, totally 40 circulations；Real-time Collection is synchronized in annealing and the extension stage of each circulation
Fluorescence, according to standard curve, converses the content of yellow twig cause of disease in testing sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410744410.7A CN104404158B (en) | 2014-12-09 | 2014-12-09 | A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410744410.7A CN104404158B (en) | 2014-12-09 | 2014-12-09 | A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104404158A CN104404158A (en) | 2015-03-11 |
| CN104404158B true CN104404158B (en) | 2016-09-28 |
Family
ID=52641821
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410744410.7A Active CN104404158B (en) | 2014-12-09 | 2014-12-09 | A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104404158B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105830749A (en) * | 2016-04-01 | 2016-08-10 | 福建省农业科学院果树研究所 | Method for screening citrus HLB (Huanglongbing) control agents through grafting |
| CN106405029A (en) * | 2016-11-15 | 2017-02-15 | 融安县植保植检站 | Cumquat epiphytic chlorococcum agentia screening test system |
| CN107047210A (en) * | 2017-05-22 | 2017-08-18 | 高必红 | A kind of prevention and controls of Citrus shatangju yellow twig |
| CN110331220B (en) * | 2019-07-23 | 2020-11-13 | 云南农业大学 | Method for screening biocontrol bacteria of citrus greening disease by two-step method |
| CN111206104B (en) * | 2019-12-20 | 2021-08-03 | 韩山师范学院 | Universal primer and method for efficiently and simply obtaining mitochondrial genome of insects in psyllium superfamily and application of universal primer and method |
-
2014
- 2014-12-09 CN CN201410744410.7A patent/CN104404158B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104404158A (en) | 2015-03-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104404158B (en) | A kind of rapid screening method of citrus yellow shoot disease prevention and control medicament | |
| CN102973542B (en) | Micromolecular substance for improving sensitivity of bacteria to antibiotics | |
| CN103385912B (en) | Pithecellobium clypearia extracts and application of extract in preparation of medicines for treating methicillin-resistant staphylococcus aureus | |
| CN101519651B (en) | Shigella flexneri phage strain and application thereof | |
| CN104586869A (en) | Small molecule metabolite for improving effect of antibiotic to eliminate pathogenic bacteria | |
| CN102824290B (en) | Liquid soap | |
| Zhao et al. | Integration of pharmacokinetic and pharmacodynamic indices of valnemulin in broiler chickens after a single intravenous and intramuscular administration | |
| Shan et al. | Pharmacokinetic/pharmacodynamic relationship of marbofloxacin against Aeromonas hydrophila in Chinese soft‐shelled turtles (Trionyx sinensis) | |
| CN103667286A (en) | Small interfering ribonucleic acid (siRNA) of schistosoma japonicum katsurada membrane-associated progesterone receptor component (PGMRC2) gene and application thereof | |
| CN102876617B (en) | Pseudomonas and purpose of pseudomonas | |
| CN102258774A (en) | Preparation method of Brucellosis live vaccine and product thereof | |
| CN108403676B (en) | Application of succinic acid in improving sensitivity of bacteria to antibiotics | |
| Kolchyk et al. | Biofilms of pathogenic bacteria in pig industry | |
| CN108294049B (en) | A kind of Paenibacillus polymyxa culture solution combination drug and preparation method thereof for treating Citrus Huanglongbing pathogen | |
| CN103141425B (en) | Producing method of sterile zebra fish | |
| CN105055539A (en) | Chinese herb extract with antibacterial effect and preparation method for Chinese herb extract | |
| CN107828866A (en) | A kind of application for the macleaya cordata conditioning agent for improving weanling pig immune performance | |
| CN104606219A (en) | Micromolecule metabolite for facilitating antibiotic to eliminate pathogenic bacteria | |
| CN104840701A (en) | Gel for treating vaginitis | |
| CN101385737A (en) | Antibiotic effective ingredient and use thereof | |
| CN114272246A (en) | Application of uracil in preparing anti-infective medicine | |
| Dhaked et al. | Antimicrobial activity of ethanolic and aqueous extract of Caesalpinia pulcherrima flowers | |
| CN103690544A (en) | Cefquinome raw material and application of cefquinome preparation to preparing avian antibacterial medicine | |
| CN104450273A (en) | Detergent for cotton/jute | |
| Chaidir et al. | Isolation and Selection of Growth Medium for Microalgae of Lake Biru Sawahlunto West Sumatra and Antibacterial Activity Test. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |