CN108513982A - AHL molecules are as the application in the disease control caused by ralstonia solanacearum of chemical pesticide sterilizing synergistic agent - Google Patents

AHL molecules are as the application in the disease control caused by ralstonia solanacearum of chemical pesticide sterilizing synergistic agent Download PDF

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CN108513982A
CN108513982A CN201810334630.0A CN201810334630A CN108513982A CN 108513982 A CN108513982 A CN 108513982A CN 201810334630 A CN201810334630 A CN 201810334630A CN 108513982 A CN108513982 A CN 108513982A
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ahl
chemical pesticide
ralstonia solanacearum
micromolecular compound
application according
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CN108513982B (en
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常长青
廖金星
刘琼光
吴熔熔
刘冉淋
章婉贤
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South China Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
    • A01N43/08—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A01N59/20—Copper
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C

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  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Environmental Sciences (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Pest Control & Pesticides (AREA)
  • Agronomy & Crop Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Inorganic Chemistry (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

A kind of synergist application the invention discloses bacteriogenic micromolecular compound as chemical pesticide fungicide, the synergist are by pseudomonas aeruginosaPseudomonas aeruginosa,PAThe 3 oxygen dodecanoyl homoserine lactones generated（AHL）, can extract or can be synthesized by artificial chemistry from P. aeruginosa fermented liquid.The micro micromolecular compound（AHL）It is used in mixed way with chemical pesticide fungicide, to ralstonia solanacearumRalstoniasolanacearumSterilization has apparent synergistic function.The method can significantly improve the bactericidal effects of chemical pesticide fungicide; the usage amount and medicament residual of chemical pesticide is greatly decreased, cost can not only be reduced, moreover it is possible to environmental protection; the increasingly serious problem of pathogen drug resistance is effectively relieved, " subtracting medicine " index and agriculture Green Development are reached in promotion.

Description

AHL molecules are anti-as chemical pesticide sterilizing synergistic agent disease caused by ralstonia solanacearum Application in controlling
Technical field
The present invention relates to pseudomonas aeruginosasPseudomonas aeruginosa,PAThe AHL small molecules of generation are as one Kind Novel synergistic agent is for chemical pesticide fungicide in ralstonia solanacearumRalstoniasolanacearumSterilization synergistic effect, Belong to biological prevention plant protection art.
Background technology
Bacterial wilt is a kind of worldwide crushing soil-borne disease, and typical systemic vascular bundle diseases, mainly Plant vasular beam necrosis is caused to be rotted, destroying plant conducting tissue finally makes whole strain wilting necrosis.Ralstonia solanacearum Ralstoniasolanacearum can disseminate peanut, potato, tobacco, eggplant, tomato, capsicum, mulberry tree Deng50Duo Ge sections number The crop of hundred kinds of Important Economics value.The disease generally occurs in subtropical zone, and the popular time often results in catastrophic loss.Mesh The prevention of preceding bacterial wilt is always to have a great difficult problem to be solved in agricultural production, does not there is the spy of special prevention bacterial wilt temporarily Imitate pesticide.
Therefore problem is prevented for bacterial wilt, researches and develops the agriculture that novel biological antibiotic synergist is added to current disease control In medicine, the preventive effect of pesticide is improved, reduces the usage amount of pesticide, indiscriminate use of pesticide problem is reduced, weakens the drug resistance of pathogen, it is right It pushes the green safe sound development of modern agriculture and safeguards that ecological safety is of great significance.Small molecule AHL of the present invention It is from pseudomonas aeruginosaPseudomonasaeruginosa,Extraction or artificial chemistry synthesis, are bacterium in PA fermentation cultures Secondary metabolites, there is no the problem of environmental pollution, while significant to solving germ resistance problems.Its structural formula is such as Under：
(Pearson, J P, Gray, K M and Passador, L, et al.,1994)
Early-stage study proves that certain density AHL and 77% can kill generate obviously ralstonia solanacearum after wettable powder mixture Sterilization synergistic effect, the usage amount of pesticide can be substantially reduced.It has not yet to see with small molecule AHL research and development into pesticide synergistic agent Relevant report.
Invention content
The technical problem to be solved by the present invention is to：A new applications of the small molecule AHL as pesticide synergistic agent is provided, is It solves the problems, such as that pesticide abuse provides a kind of effective ways, substantially reduces the usage amount of pesticide.
Technical solution provided by the invention is：A kind of synergist AHL small molecules and chemical pesticide fungicide are green withered in prevention The application of sick aspect.
The small molecule AHL is from pseudomonas aeruginosaPseudomonasaeruginosa,It is carried in PA fermentation cultures It takes or artificial chemistry synthesizes.
The extracting method of the small molecule AHL is：Centrifugation pseudomonas aeruginosa is incubated overnight liquid and obtains supernatant, supernatant first Middle addition ethyl acetate extraction, utilizes high performance liquid chromatography（HPLC）It is isolated and purified, active material is collected, through structural analysis The small molecule of pseudomonas aeruginosa generation is confirmed as in identification.
Above-mentioned application, the chemical agent for preventing plant disease caused by ralstonia solanacearum is can to kill to obtain wettable powder Agent.
Above-mentioned application is by the chemical agent of micromolecular compound AHL and plant disease caused by prevention ralstonia solanacearum Mixture uses, to increase control effect.
Above-mentioned application, when mixture, a concentration of 40uM ~ 200uM of the micromolecular compound AHL, preferably for 100uM~200uM；In a specific embodiment, a concentration of 100uM, 150uM of the micromolecular compound AHL, or 200uM。
The compounding method is：Small molecule AHL obtained by separation and Extraction is configured to different concentration gradient solution（Phase The same volume 5ul signaling molecule methanol solutions of AHL containing various concentration）, respectively on chemical pesticide mixed processing synthetic medium Bacterial wilt opportunistic pathogen records colony counts and growing state.
Test result shows that certain density small molecule AHL can significantly increase chemical pesticide and be killed to bacterial wilt opportunistic pathogen Bacterium ability has using small molecule AHL as a kind of new and effective sterilization pesticide synergist in terms of preventing agricultural plant disease There are very high utility value and development prospect.
Production application cost after taking into consideration and bactericidal effect, 100 μM ~ 200 μM small molecule AHL's of present invention selection Concentration range with chemical pesticide mixed preparing as preventing the preferred concentration range of bacterial wilt.
The invention has the advantages that：The present invention sets about from a new direction of exploitation sterilizing synergistic agent, using thin The signaling molecule AHL that bacterium itself generates and chemical pesticide mixture, the results showed that the drug effect of chemical pesticide fungicide can be significantly improved, The usage amount and medicament residual of fungicide is greatly reduced, cost can not only be reduced, and itself be the natural products pair of bacterium It is more environment-friendly, moreover it is possible to which that the increasingly serious problem of pathogen drug resistance is effectively relieved in environmental protection, and " subtracting medicine " index is reached in promotion With agriculture Green Development, it is of great significance to the solution of severe pathogen resistance problems.Present invention determine that small molecule AHL prevents applicable a concentration of 100uM ~ 200uM of bacterial wilt as the synergist of chemical pesticide fungicide.
Description of the drawings
Fig. 1 small molecule AHL mixtures 77% can kill wettable powder to ralstonia solanacearum sterilize synergistic effect（It is dilute to handle bacterium solution Release 10 times）, wherein 1A：WT（It is not added with any substance processing）；1B：Phe（Individually with agricultural 77% can kill at wettable powder Reason）；1C：Agricultural 77% can kill wettable powder dilution agent 200 again with 40 μM of AHL mixtures；1D：Agricultural 77% can kill to obtain wettable Dilution for powder 200 again with 80 μM of AHL mixtures；1E：Agricultural 77% can kill wettable powder dilution agent 200 is mixed with 100 μM of AHL again Match；1F：Agricultural 77% can kill wettable powder dilution agent 200 again with 150 μM of AHL mixtures；1G：Agricultural 77% can kill to obtain wettable Dilution for powder 200 again with 200 μM of AHL mixtures；Note：Treatment fluid dilution 104Times coated plate.
Fig. 2 small molecules AHL mixtures agricultural 77% can kill wettable powder to ralstonia solanacearum bactericidal effect CFU statistical results, Phe is that individually can kill to obtain wettable powder processing with agricultural 77%, and it is mixed that P-40 ~ P-200 is expressed as 40 μM ~ 200 μM AHL of selection Wettable powder processing bacterium solution can be killed to obtain with agricultural 77%.
Fig. 3 small molecules AHL mixtures agricultural 77% can kill wettable powder to ralstonia solanacearum sterilization synergistic effect statistics knot Fruit, wherein：P-40 ~ P-200, which is expressed as 40 μM ~ 200 μM AHL mixtures agricultural 77% of selection, can kill to obtain wettable powder processing Bacterium solution；Various concentration AHL as synergist mixture agricultural 77% can kill synergy ratio=agricultural 77% of wettable powder can kill CFU/ is applied alone in wettable powder（Agricultural 77% can kill to obtain wettable powder+AHL synergist）CFU.
0.5% methanol of Fig. 4 is to ralstonia solanacearum influence, 4A:WT (individually plus the sterile water process of 5ul)；4B (individually adds 5ul methyl alcohol process）.
Specific implementation mode
By the following examples with test example present invention be described in more detail content, but present disclosure not only office It is limited to the following examples, is suitable for various chemical pesticide fungicide
The preparation of 1 AHL small molecules of embodiment
By the pseudomonas aeruginosa seed liquor being incubated overnight access YEB culture mediums, 28oC, after 200rpm cultivates 24 hours, lead to It crosses and culture solution supernatant is collected by centrifugation.Gained supernatant small molecule to be extracted is used.Wherein, YEB culture medium prescriptions are：0.5 g/L MgSO4•7H2O, 5 g/L Sucrose, 5 g/L Yeast Extract, 10g/L Trypton, 5 g/L NaCl, pH 7.0。
The present invention using conventional compound separation and Extraction method to the separation of active material in zymotic fluid with carry It takes.After culture solution supernatant is collected by centrifugation, addition and the isometric ethyl acetate of supernatant are put into after 4 DEG C of standing 2h in 20 DEG C Shaking table handles 1h, is extracted with separatory funnel, collects upper layer liquid separation.Upper layer liquid separation revolving of the Rotary Evaporators by collection is recycled, After organic solvent is evaporated, eluted using 2-3mL methanol.Methanol solution containing active constituent is evaporated concentration and then profit Use high performance liquid chromatography（HPLC）It is further purified, analyzes, collects active material.Structural Identification confirms that active constituent is AHL.
Embodiment 2 small molecule AHL and 77% can kill to obtain sterilization synergistic effect of the wettable powder mixture to ralstonia solanacearum
Using bacterium colony counting statistics method（CFU methods）Wettable powder can be killed to obtain by measuring various concentration small molecule AHL extracts pair 77% Sterilization synergistic effect：It uses methanol to configure small molecule AHL as solvent and is diluted into certain density medicament, take overnight training It supports to OD600=1.0 ralstonia solanacearum bacterium solution, wettable powder, setting one can be killed to obtain by being added the 77% of 800 times of 100mg/ml of dilution It is a to be not added with small molecule AHL processing as positive control, then a series of tool concentration gradients are set（40μM 、80μM 、100μM、150μ M、200μM）Small molecule AHL can kill to obtain the processing that mixes of wettable powder with 77%.All processing are 28oC, 200rpm is cultivated After 24 hours, dilution 104Times, therefrom take 200 μ L bacterium solution coated plates（Culture medium is LB）, it is placed in 28 DEG C of constant incubators and trains It supports.Observation and statistical result after about 3 ~ 4 days.
Statistics flat-plate bacterial colony number can obtain：Individually with 77% can kill wettable powder processing CFU be 1.87 × 104, use 77% can kill the CFU that is handled again with 40 μM of AHL mixtures of wettable powder dilution agent 800 is 1.64 × 104, with 77% can kill can It is 1.38 × 10 that wet powder, which dilutes 800 times and the CFU of 80 μM of AHL mixtures processing,3, with 77% can kill to obtain wettable powder dilution agent 800 times and 100 μM of AHL mixtures processing CFU be 7.47 × 103, with 77% can kill wettable powder dilution agent 800 again with 150 μ The CFU of M AHL mixtures processing is 3.77 × 103, with 77% can kill wettable powder dilution agent 800 again at 200 μM of AHL mixtures The CFU of reason is 7.4 × 102（Refer to Fig. 1 and Fig. 2）.With 40 μM of small molecule AHL to 77% can kill wettable powder kills Bacterium synergy ratio is 1.15 times, with 80 μM of AHL pairs 77% of small molecule can kill the sterilization synergy ratio of wettable powder is 1.35 Times, with 100 μM of AHL pairs 77% of small molecule can kill wettable powder sterilization synergy ratio be 2.18 times, it is small with 150 μM AHL pairs 77% of molecule can kill wettable powder sterilization synergy ratio be 2.62 times, can with 200 μM of AHL pairs 77% of small molecule Kill wettable powder sterilization synergy ratio be 5.65 times（Refer to Fig. 3）.0.5% methanol has no significant effect ralstonia solanacearum （Refer to Fig. 4）.

Claims (8)

1. applications of the micromolecular compound AHL as the synergist of the chemical agent of plant disease caused by prevention ralstonia solanacearum.
2. application according to claim 1, it is characterised in that：The ralstonia solanacearum can cause plant to cause bacterial wilt, special It is other that most serious, such as tomato, potato, tobacco are endangered to plant of Solanaceae.
3. application according to claim 1, it is characterised in that：The micromolecular compound AHL is from pseudomonas aeruginosa Extraction obtains in zymotic fluid or artificial chemistry synthesizes.
4. application according to claim 1, which is characterized in that the chemistry of plant disease caused by the prevention ralstonia solanacearum Medicament is can to kill to obtain wettable powder.
5. application according to any one of claims 1 to 4, which is characterized in that be that micromolecular compound AHL is green with prevention The chemical agent mixture of the microbial plant disease of blight uses, to increase control effect.
6. application according to claim 5, which is characterized in that when mixture, the micromolecular compound AHL's is a concentration of 40uM~200uM。
7. application according to claim 6, which is characterized in that when mixture, the micromolecular compound AHL's is a concentration of 100uM~200uM。
8. application according to claim 7, which is characterized in that when mixture, the micromolecular compound AHL's is a concentration of 100uM, 150uM or 200uM.
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CN112029789A (en) * 2020-08-03 2020-12-04 华南农业大学 Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage
CN114317319A (en) * 2021-11-10 2022-04-12 福建省农业科学院植物保护研究所 Pseudomonas aeruginosa and metabolite and application thereof

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CN112029789A (en) * 2020-08-03 2020-12-04 华南农业大学 Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage
CN112029789B (en) * 2020-08-03 2022-05-31 华南农业大学 Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage
CN114317319A (en) * 2021-11-10 2022-04-12 福建省农业科学院植物保护研究所 Pseudomonas aeruginosa and metabolite and application thereof
CN114317319B (en) * 2021-11-10 2023-04-28 福建省农业科学院植物保护研究所 Pseudomonas aeruginosa and metabolite and application thereof

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