CN104404158A - Method for rapidly screening controlling agrochemicals for citrus greening disease - Google Patents
Method for rapidly screening controlling agrochemicals for citrus greening disease Download PDFInfo
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Abstract
The invention belongs to the technology of screening of a chemical agent for prevention and control in the agricultural field, and particularly discloses a method for rapidly screening controlling agrochemicals for a citrus greening disease. The method uses diaphorina citri carrying bacteria as an initial evaluation material, and adopts RT-qPCR to monitor the growth and decline level of citrus greening disease pathogenic bacteria in the diaphorina citri carrying bacteria, so as to evaluate the pesticide effect. The preparation method creatively takes the diaphorina citri carrying bacteria as an screening material of a chemical agent for being resistant to the citrus greening disease pathogenic bacteria, can rapidly screen and evaluate a large amount of controlling agrochemicals for citrus greening disease within a short time, solves the deficiencies in the existing chemical agent for being resistant to the citrus greening disease pathogenic bacteria that the screening period is too long, and a set of accurate and effective evaluation system is absent.
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 rapid screening method of citrus yellow shoot disease prevention and control medicament.
Background technology
Citrus yellow shoot disease occurs with a long history in China, endangers day by day serious.Have made be Reinking in 1919 in Chao-Shan Area, China Guangdong Late Cambrian citrus yellow shoot disease.To 20th century the mid-80, citrus yellow shoot disease in China Guangdong, Guangxi, Fujian, Hainan and Taiwan citrus producing region extensive widespread, and in Zhejiang, Guizhou, the part citrus producing region, ground such as Hunan occur in succession.There are 19 of citrus culture provinces in China, existing 11 of district is endangered, and injured area accounts for more than 80% of the total cultivated area of citrus, about 85% (Fan Guocheng etc., 2009) of ultimate production.But, in view of yellow twig germ ('
candidatusliberibacter asiaticus ', is called for short Las) difficult characteristic of cultivating, relative chemical agent screening operation is still made slow progress so far.
The anti-yellow twig germ chemical agent screening experiment carried out for citrus yellow shoot disease chemical prevention.At present, be at home and abroad also in progress all to some extent.But, early stage screening method is generally based on the symptom variation for morbidity plant itself, often lack a set of accurate and effective appraisement system, and also exist the screening cycle longer (generally need 1-3 even longer time continuous monitoring), be easily affected by the external environment wait deficiency.As: trunk injection apparatus, phloem method of topical application, foliar spray method and soil drench etc.Until in recent years, in view of the harm situation day of citrus yellow shoot disease is severeer, and present stage yellow twig germ fail always and carry out separation and Culture, just make more scholar efforts be made so that by other disease sources of citrus yellow shoot disease screening and assessment system as its pesticide control.As Muqing Zhang(2010,2011) etc. utilize the surviving rate of Vinca branch after chemicals treatment having infected yellow twig to carry out evaluate efficacy, construct the yellow twig chemical prevention medicament primary dcreening operation system based on Vinca.Though this primary dcreening operation system substantially reduces screening time, but still need at least 60-90 d could obtain preliminary the selection result.Generally speaking, current anti-yellow twig germ chemical agent screening excessive cycle, and lack a set of accurate and effective appraisement system.
The yellow twig chemical prevention medicament primary dcreening operation system based on Vinca is constructed in prior art, utilize PCR detection technique, with the content of the regeneration biological amount that survives of 2 ~ 6 months Vincas and the Candidatus liberibacter of newborn long shoot bar after Chemical treatment for index, filter out the medicine of anti-Candidatus liberibacter, screening time is long, may there is the inaccurate situation of screening.And the present invention creatively utilizes the screening material of citrus psylla as anti-yellow twig germ chemical agent that carry disease germs, and adopt yellow twig germ described in RT-qPCR real-time dynamic monitoring in the growth and decline degree in citrus psylla body of carrying disease germs, only need the chemical agent that can filter out effective anti-citrus yellow shoot disease germ for 5 days, screening time is short, accuracy is high, provide one anti-yellow twig germ chemical agent screening method more fast and effectively, solve current anti-yellow twig germ chemical agent screening excessive cycle, lack the deficiencies such as a set of accurate and effective appraisement system.
Preferably, described method comprises the steps:
S1. to feed process: after the appropriate chemical agent solution of the citrus psylla imago feeding that carries disease germs, then raise by healthy citrus leaves;
S2. evaluating drug effect: extract the citrus psylla STb gene that carries disease germs after process of feeding, then carry out preliminary assessment drug effect by the growth and decline degree of RT-qPCR technology real-time dynamic monitoring yellow twig germ in wood louse body.
Preferably, the concentration of described chemical agent solution is 1000 mg/L.
Preferably, the treatment 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, and 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, the upstream and downstream primer of 10 μm of ol/L and each 0.4 μ L of probe, DNA profiling 1 μ L, then adds sterilizing ddH
2o to cumulative volume 20 μ L, reaction conditions is after 95 DEG C of denaturation 30 s; 95 DEG C of annealing 5 s, 60 DEG C extend 30 s, totally 40 circulations; At annealing and the extension stage synchronous Real-time Collection fluorescence of each circulation, according to typical curve, converse the content of yellow twig cause of disease in testing sample.
The present invention found through experiments, and after 5d process, filters out the tetracycline hydrochloride in tetracycline antibiotics and tetramycin hydrochloride, and the gentamicin sulphate in aminoglycoside antibiotics and amikacin sulfate are the chemical agent of effective anti-citrus yellow shoot disease germ.
The present invention also provides a kind of application of rapid screening method of Anti-bacterium chemical agent, the evaluating material of described method is the disease source insect of its germ, and adopt germ described in RT-qPCR real-time dynamic monitoring to carry out evaluate efficacy in the growth and decline degree in insect body of carrying disease germs, described method is applied to the screening of the difficulty cultivation germ chemical prevention medicament that sucking pest is propagated.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention creatively utilizes the screening material of citrus psylla as anti-yellow twig germ chemical agent that carry disease germs, rapid screening and evaluation can be carried out at short notice to citrus yellow shoot disease prevention and control medicament, solve current anti-yellow twig germ chemical agent screening excessive cycle, lack the deficiencies such as a set of accurate and effective appraisement system.
(2) the present invention adopts the preliminary assessment material of citrus psylla as anti-yellow twig germ chemical agent that carry disease germs, evaluate efficacy is carried out by the growth and decline degree of Real-Time Fluorescent Quantitative PCR Technique (RT-qPCR) real-time dynamic monitoring yellow twig germ in its body, only need the chemical agent that can filter out effective anti-citrus yellow shoot disease germ for 5 days, provide one more quick, effective anti-yellow twig germ chemical agent screening method, screen the efficient citrus yellow shoot disease prevention and control medicament obtained, both the citrus yellow shoot disease chemical prevention medicament that can be used as in production is directly utilized, also can be the research and development preventing and treating citrus yellow shoot disease pharmaceutical preparations, and citrus yellow shoot disease germ and host do the research work such as mechanism mutually and provide theoretical reference.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of rapid screening method of citrus yellow shoot disease prevention and control medicament.The present invention creatively utilizes the screening material of citrus psylla as anti-yellow twig germ chemical agent that carry disease germs, rapid screening and evaluation can be carried out at short notice to citrus yellow shoot disease prevention and control medicament, solve current anti-yellow twig germ chemical agent screening excessive cycle, lack the deficiencies such as a set of accurate and effective appraisement system.
Object 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 material of described citrus yellow shoot disease prevention and control medicament is the citrus psylla that carries disease germs, and adopts yellow twig germ described in RT-qPCR real-time dynamic monitoring to carry out evaluate efficacy in the growth and decline degree in citrus psylla body of carrying disease germs.
accompanying drawing explanation
Fig. 1 is the impact of different time process on yellow twig germ concentration in citrus psylla body.
Fig. 2 is the Antibacterial Activity result of tetracycline antibiotics to yellow twig germ in citrus psylla body.
Fig. 3 is the Antibacterial Activity result of aminoglycoside antibiotics to yellow twig germ in citrus psylla body.
Fig. 4 is the Antibacterial Activity result of β-lactam antibitics to yellow twig germ in citrus psylla body.
Fig. 5 is that Comprecin and other antimicrobial drugs are to the Antibacterial Activity result of yellow twig germ in citrus psylla body.
Fig. 6 is that tetracycline hydrochloride and tetramycin hydrochloride are to the removal effect of yellow twig germ in citrus psylla body.
Caption (Fig. 1-3): CK represents blank group; * warp is represented
tinspection exist significant difference (
p< 0.05); * represents warp
tinspection exist extremely significant difference (
p< 0.01).
embodiment
Below in conjunction with Figure of description and specific embodiment, the present invention is described in further details, but embodiment does not limit in any form the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.
The specific embodiment of the invention is mainly used in the rapid screening of anti-yellow twig germ chemical agent, and suitable change is for examination insect, chemical agent kind and feeding device, the method for the invention can also be applied to the screening of the difficulty cultivation germ chemical prevention medicament that other suckings pest are propagated.
embodiment 1the rapid screening process of anti-yellow twig germ chemical agent
Get the citrus psylla that carries disease germs after 15 hunger 12 h, with feeding 24h for examination chemical agent solution of 3 mL 1000 mg/L, 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 the moisturizing situation of once healthy citrus leaves every 24 h, and add appropriate corresponding chemical liquid medicine simultaneously and carry out moisturizing process, the citrus psylla that causes to prevent healthy citrus leaves excessive dehydration carrying disease germs cannot normally take food.Not add the distilled water of any chemical agent for blank, each process all arranges 3 repetitions.Chemical agent is fed after process completes, and adopts OMEGA
?e.Z.N.A. Insect DNA Kit test kit (D0926-01) that company buys extracts the citrus psylla STb gene that carries disease germs, and the growth and decline degree of recycling RT-qPCR technology real-time dynamic monitoring yellow twig germ in its body carrys out preliminary assessment drug effect.
The RT-qPCR of Las detects primer HLBasf/HLBasr(5 '-TCG AGCGCGTATGCAATACG-3 '/5 '-GCGTTATCCCGTAGAAAAAGGTAG-3 ' reported with reference to (2006) such as Li), its nucleotide sequence is respectively as shown in SEQ ID NO:1, SEQ ID NO:2; Probe HLBp(5 '-FAM-AGACGGGTGAGTAACGCG-BHQ-3 '), its nucleotide sequence probe and primer as shown in SEQ ID NO:3 synthesize by the precious biotechnology company limited in Dalian.RT-qPCR reaction system is: 2 × TaqMan PCR Master Mix 10 μ L, the upstream and downstream primer of 10 μm of ol/L and each 0.4 μ L of probe, DNA profiling 1 μ L, then adds sterilizing ddH
2o to cumulative volume 20 μ L.Often wheel reaction all arranges the standard substance (A4 plasmid) of 7 gradients as outer ginseng drawing standard curve, ddH
2o is blank, and healthy citrus DNA sample is negative control, and experiment repetition more than 3 times.Reaction is carried out in CFX96 Touch quantitative fluorescent PCR detection system, and reaction conditions is: after 95 DEG C of denaturation 30 s, 40 circulation (95 DEG C of annealing 5 s; 60 DEG C, extend 30 s), at annealing and the extension stage synchronous Real-time Collection fluorescence of each circulation.Programming and result read and all use Bio-Rad CFX Manger 2.1 software.According to the typical curve that A4 plasmid is joined, converse the content of yellow twig cause of disease in testing sample outward.
embodiment 2different time process is on the impact of yellow twig germ concentration in citrus psylla body
The different treatment time of research tetracycline hydrochloride is on the impact of the fungistatic effect of yellow twig germ in the citrus psylla body that carries disease germs.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing tetracycline hydrochloride, 1,3,5,7 and 9 d are raised respectively again by healthy citrus leaves, experimental result is shown in Fig. 1, and in citrus psylla body, the inhibiting rate of yellow twig germ concentration is respectively: 14.19%, 25.54%, 59.17%, 68.88% and 89.10%; In blank group citrus psylla body, the concentration of yellow twig germ then rises-1.55%, 25.51%, 29.21%, 39.89% and 37.86% respectively.And after 3 d process, in treatment group citrus psylla body, between the inhibiting rate of yellow twig germ concentration and blank group, there is significant difference (P<0.05); After 5,7 and 9 d process, in treatment group citrus psylla body, between the inhibiting rate of yellow twig germ concentration and blank group, there is pole significant difference (P<0.01).Meanwhile, find the change of yellow twig germ concentration in citrus psylla body 3 d and 5 d process place the most obvious.Therefore, preliminary selected 5 d are its optimum handling number of days.Thereafter, other all chemical agent solution are all by 5 d process.
embodiment 3tetracycline antibiotics is on the impact of the bacteriostatic activity of yellow twig germ in the citrus psylla body that carries disease germs
Research tetracycline antibiotics is on the impact of the bacteriostatic activity of yellow twig germ 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 5 d by healthy citrus leaves, in its body, the inhibiting rate of yellow twig germ concentration is 5.58%, with there was no significant difference before process; After the citrus psylla that carries disease germs takes food the chemical agent solution containing tetracycline hydrochloride or tetramycin hydrochloride respectively, in its body, the inhibiting rate of yellow twig germ concentration is respectively 59.17% and 49.70%, and with process before there is pole significant difference (P<0.01).But when after the chemical agent solution that the citrus psylla that carries disease germs takes food containing Doxycycline Hyclate, in its body, yellow twig germ concentration but rises 34.90%.This shows, the tetracycline hydrochloride in tetracycline antibiotics and tetramycin hydrochloride all can effectively suppress yellow twig germ at citrus psylla proliferation in vivo, and extremely significantly can reduce the yellow twig germ concentration of carrying disease germs in citrus psylla body; Though hydrochloric acid Klinomycin also can suppress yellow twig germ at citrus psylla proliferation in vivo to a certain extent, it is not obvious to the reducing effect of yellow twig germ concentration in the citrus psylla body that carries disease germs; And Doxycycline Hyclate effectively can not suppress the propagation of yellow twig germ in citrus psylla body.Result shows, tetracycline hydrochloride and tetramycin hydrochloride are screen tetracycline antibiotics used from this effective anti-citrus yellow shoot disease germ chemical agent filtered out.
embodiment 4aminoglycoside antibiotics is on the impact of the bacteriostatic activity of yellow twig germ in citrus psylla body
Research aminoglycoside antibiotics is on the impact of the bacteriostatic activity of yellow twig germ in citrus psylla body, and experimental result is shown in Fig. 3.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing Vetstrep, then raise 5 d by healthy citrus leaves, in its body, the inhibiting rate of yellow twig germ concentration is 6.97%; And after taking food the chemical agent solution containing sulphuric acid kanamycin, in its body, the concentration of yellow twig germ then rises 6.22%, but all with process before there was no significant difference.After the citrus psylla that carries disease germs takes food the chemical agent solution containing gentamicin sulphate or amikacin sulfate respectively, in its body, the inhibiting rate of yellow twig germ concentration is respectively 41.21% and 42.69%, and with process before there is pole significant difference (P<0.01).This shows, the gentamicin sulphate in aminoglycoside antibiotics and amikacin sulfate all can effectively suppress yellow twig germ at citrus psylla proliferation in vivo, and extremely significantly can reduce the yellow twig germ concentration of carrying disease germs in citrus psylla body; And though Vetstrep also can suppress yellow twig germ at citrus psylla proliferation in vivo to a certain extent, it is not obvious to the reducing effect of yellow twig germ concentration in the citrus psylla body that carries disease germs; And sulphuric acid kanamycin effectively can not suppress the propagation of yellow twig germ in citrus psylla body.Result shows, gentamicin sulphate and amikacin sulfate are screen aminoglycoside antibiotics used from this effective anti-citrus yellow shoot disease germ chemical agent filtered out.
embodiment 5β-lactam antibitics is on the impact of the bacteriostatic activity of yellow twig germ in citrus psylla body
Research β-lactam antibitics is on the impact of the bacteriostatic activity of yellow twig germ in citrus psylla body, and experimental result is shown in Fig. 4.After the citrus psylla that carries disease germs takes food chemical agent solution 24 h containing Ampicillin Trihydrate or ceftazime respectively, 5 d are raised respectively again by healthy citrus leaves, in its body, the inhibiting rate of yellow twig germ concentration is respectively 7.40% and 21.71%, but all front without significance sex differernce with process; When after the chemical agent solution that the citrus psylla that carries disease germs takes food containing penicillin G, in its body, yellow twig germ concentration but rises 39.60%.This shows, though Ampicillin Trihydrate in β-lactam antibitics and ceftazime can suppress yellow twig germ at citrus psylla proliferation in vivo to a certain extent, not obvious to the reducing effect of yellow twig germ concentration in the citrus psylla body that carries disease germs; And penicillin G effectively can not suppress the propagation of yellow twig germ in citrus psylla body.Result shows, this screening is not yet screened β-lactam antibitics used from this, filters out effective anti-citrus yellow shoot disease germ chemical agent.
embodiment 6comprecins etc. are on the impact of the bacteriostatic activity of yellow twig germ in citrus psylla body
The impact on the bacteriostatic activity of yellow twig germ in citrus psylla body such as research Comprecin, experimental result is shown in Fig. 5.When the citrus psylla that carries disease germs takes food after respectively containing chemical agent solution 24 h of ciprofloxacin HCl, Pefloxacin, soluble chloride mycin or benziothiazolinone, raise 5 d respectively by healthy citrus leaves again, in its body, yellow twig germ concentration rises 12.37%, 30.84%, 13.02% and 26.13% respectively.This shows, the ciprofloxacin HCl in Comprecin, Pefloxacin, and the soluble chloride mycin in chloromycetin series antibiotics and benziothiazolinone etc. all effectively can not suppress the propagation of yellow twig germ in citrus psylla body.Result shows, this screening is not yet screened Comprecin used and chloromycetin series antibiotics from this, filters out effective anti-citrus yellow shoot disease germ chemical agent.
embodiment 7tetracycline hydrochloride and tetramycin hydrochloride are to the removal effect of yellow twig germ in citrus psylla body
Research tetracycline hydrochloride and tetramycin hydrochloride are to the removal effect of yellow twig germ in citrus psylla body, and experimental result is shown in Fig. 6.When after chemical agent solution 24 h that the citrus psylla that carries disease germs takes food containing tetracycline hydrochloride, raise 15 d(respectively by healthy citrus leaves again and survive 8) and 20 d(survive 3), in its body, the inhibiting rate of yellow twig germ concentration is respectively 98.36% and 100.00%; And after the citrus psylla that carries disease germs takes food chemical agent solution 24 h containing tetramycin hydrochloride, raise 15 d(respectively by healthy citrus leaves again and survive 11) and 20 d(survive 5), in its body, the inhibiting rate of yellow twig germ concentration is respectively 95.87% and 99.29%.Experimental result tentatively shows, the yellow twig germ carried disease germs in citrus psylla body, can be removed by the chemical agent solution taken food for a long time containing tetracycline hydrochloride or tetramycin hydrochloride.
SEQUENCE LISTING
<110> Agricultural University Of South China
The rapid screening method of a <120> 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 (6)
1. the rapid screening method of a citrus yellow shoot disease prevention and control medicament, it is characterized in that, 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 adopts yellow twig germ described in RT-qPCR real-time dynamic monitoring to carry out evaluate efficacy in the growth and decline degree in citrus psylla body of carrying disease germs.
2. method according to claim 1, is characterized in that, comprises the steps:
S1. to feed process: after the appropriate chemical agent solution of the citrus psylla imago feeding that carries disease germs, then raise by healthy citrus leaves;
S2. evaluating drug effect: extract the citrus psylla STb gene that carries disease germs after process of feeding, then carry out preliminary assessment drug effect by the growth and decline degree of RT-qPCR technology real-time dynamic monitoring yellow twig germ in wood louse body.
3. method according to claim 2, is characterized in that, the concentration of described chemical agent solution is 1000 mg/L.
4. method according to claim 3, is characterized in that, the treatment time of described chemical agent is 24h.
5. method according to claim 4, is characterized in that, described wood louse adult is used further to S1 step after Nature enemy 12h.
6. method according to claim 5, is characterized in that, in described S2 step, the detection primer of RT-qPCR real-time dynamic monitoring is HLBasf/HLBasr, and 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, the upstream and downstream primer of 10 μm of ol/L and each 0.4 μ L of probe, DNA profiling 1 μ L, then adds sterilizing ddH
2o to cumulative volume 20 μ L, reaction conditions is after 95 DEG C of denaturation 30 s; 95 DEG C of annealing 5 s, 60 DEG C extend 30 s, totally 40 circulations; At annealing and the extension stage synchronous Real-time Collection fluorescence of each circulation, according to typical curve, converse the content of yellow twig cause of disease in testing sample.
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Cited By (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 |
| CN110331220A (en) * | 2019-07-23 | 2019-10-15 | 云南农业大学 | The method of two-step method screening Citrus Huanglongbing pathogen biocontrol microorganisms |
| CN111206104A (en) * | 2019-12-20 | 2020-05-29 | 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
Non-Patent Citations (2)
| Title |
|---|
| WENBIN LI等: "Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing", 《JOURNAL OF MICROBIOLOGICAL METHODS》, vol. 66, 18 January 2006 (2006-01-18), pages 104 - 115 * |
| 陈仕钦等: "柑橘黄龙病防控药剂筛选试验初报", 《植物保护》, vol. 40, no. 2, 1 August 2014 (2014-08-01), pages 166 - 170 * |
Cited By (6)
| 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 |
| CN110331220A (en) * | 2019-07-23 | 2019-10-15 | 云南农业大学 | The method of two-step method screening Citrus Huanglongbing pathogen biocontrol microorganisms |
| 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 |
| CN111206104A (en) * | 2019-12-20 | 2020-05-29 | 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 |
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| Publication number | Publication date |
|---|---|
| CN104404158B (en) | 2016-09-28 |
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