CN108576018B - Application of AHL molecule as chemical pesticide sterilization synergist in disease control caused by Dieckea zeae - Google Patents
Application of AHL molecule as chemical pesticide sterilization synergist in disease control caused by Dieckea zeae Download PDFInfo
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- CN108576018B CN108576018B CN201810334629.8A CN201810334629A CN108576018B CN 108576018 B CN108576018 B CN 108576018B CN 201810334629 A CN201810334629 A CN 201810334629A CN 108576018 B CN108576018 B CN 108576018B
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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
- A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
- A01N43/82—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with three ring hetero atoms
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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
Abstract
The invention discloses application of a small molecular compound generated by bacteria as a synergist of a chemical pesticide bactericide, wherein the synergist is prepared from pseudomonas aeruginosaPseudomonas aeruginosa,PAThe produced 3-oxygen lauroyl homoserine lactone (AHL) can be extracted from pseudomonas aeruginosa fermentation liquor or can be artificially and chemically synthesized. The small molecular compound (AHL) is mixed with pesticide bactericide for treating Dieckea zeaeErwiniachrysanthemipv.ZeaeThe prevention and the treatment of the caused diseases have obvious synergistic effect. The invention can obviously improve the sterilization effect of the chemical pesticide bactericide, greatly reduce the use amount of the chemical pesticide and the medicament residue, not only reduce the cost, but also protect the environment, effectively relieve the increasingly severe problem of the drug resistance of pathogenic bacteria, and promote the achievement of the 'drug reduction' index and the green agricultural development.
Description
Technical Field
The invention relates to pseudomonas aeruginosaPseudomonas aeruginosa,PAThe produced AHL micromolecule is used as a novel synergist for chemical pesticide bactericide to Dieckea zeaeErwiniachrysanthemipv. ZeaeThe sterilization and synergism, belonging to the field of plant protection in biological control technology.
Background
Dieckea zeaeErwiniachrysanthemipv.ZeaeThe plant pathogenic bacteria are plant pathogenic bacteria of worldwide importance, have wide host range, can infect 21 plants of 14 families, such as Araceae, Crassulaceae, Cactaceae, Euphorbiaceae, Liliaceae, Sonchus, Caryophyllaceae, Musaceae, Asteraceae, Compositae, Lepidaceae, Caiacaceae, Cruciferae, Umbelliferae and the like, mainly cause soft rot, and seriously affect the development of agricultural industry in China. WhereinD. zeaeThe caused bacterial basal rot of rice and bacterial soft rot of banana seriously threaten the planting of rice in rice growing areas and banana producing areas. The bacterial basal rot of rice is one of the important bacterial diseases on rice, which causes a great reduction of yield of rice. The soft rot of banana, which causes the rotten and lodging of pseudostem, is a serious banana disease newly generated in recent years, and has a tendency of serious year by year, thus bringing huge economic loss to the banana industry. The pathogen has the characteristics of quick transmission, strong destructiveness, long soil persistence time and the like. At present, the related prevention and treatment measures of the disease are single, and chemical pesticides are mainly used.
The use of pesticides, particularly abuse, causes a series of environmental problems, threats to human and animal health, induces pathogenic bacteria to generate drug resistance and the like. Therefore, how to improve the control effect of the pesticide and reduce the using amount of the pesticide is an urgent problem to be solved for promoting the green, safe and healthy development of modern agriculture and maintaining the stability of an ecosystem.
The application of the pesticide synergist can obviously reduce the usage amount of chemical pesticides. The traditional synergist improves the sterilization effect of the pesticide mainly by improving the physicochemical properties of the pesticide, such as wetting, permeability and the like, but the synthesis route is long and pollutes the environment. The novel biological sterilization synergist is developed to inhibit the detoxification of pathogenic bacteria to pesticides and weaken the drug resistance of the pathogenic bacteria, so that the control effect of the pesticides is improved, and considerable economic benefit and application prospect are achieved.The small molecule AHL of the invention is prepared from pseudomonas aeruginosaPseudomonas aeruginosa,PAThe fermentation culture solution is extracted or artificially and chemically synthesized, is a secondary metabolite of bacteria, has no problem of environmental pollution, and has important significance for solving the problem of drug resistance of pathogenic bacteria. The structural formula is as follows:
(Pearson, J P, Gray, K M and Passador, L, et al.,1994)
earlier researches prove that after AHL with a certain concentration and thiabendazole copper are mixed, obvious sterilization and synergism can be generated on the Dieckea zeae, and the using amount of pesticide can be obviously reduced. At present, no report related to the development of a pesticide synergist from a small molecular AHL exists.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a new application of the micromolecule AHL as a pesticide synergist, provides an effective method for solving the problem of pesticide abuse, and obviously reduces the using amount of pesticide.
The technical scheme provided by the invention is as follows: the application of the small molecular compound AHL as a synergist of a chemical agent for preventing and treating plant diseases caused by the Dieckea zeae.
The plant disease is soft rot.
The plant disease is rice bacterial basal rot or banana bacterial soft rot.
The chemical agent for preventing and treating the plant diseases caused by the Dieckea zeae is Thiodiazole.
The chemical agent is 20% of thiediazole copper, and the using concentration of the chemical agent is 500 times of that of a diluent.
The small molecule AHL is prepared from pseudomonas aeruginosaPseudomonas aeruginosa,PAExtracting in fermentation culture solution or artificially chemically synthesizing.
The extraction method of the small molecular AHL comprises the following steps: firstly, centrifuging the culture solution of pseudomonas aeruginosa overnight to obtain a supernatant, adding ethyl acetate into the supernatant for extraction, separating and purifying by using High Performance Liquid Chromatography (HPLC), collecting active substances, and identifying by structural analysis to determine small molecules generated by the pseudomonas aeruginosa.
The application is that the small molecular compound AHL and a chemical agent for preventing and treating plant diseases caused by the Dieckea zeae are mixed for use, so that the prevention and treatment effect is improved.
The mixing method comprises the following steps: preparing different concentration gradient solutions (5 ul of methanol solution containing different concentration AHL signal molecules with the same volume) from separated and extracted small molecular AHL, and mixing with chemical pesticide respectively to treat pathogenic bacteria on artificial culture mediumEC1The number of colonies and growth were recorded.
Test results show that the micromolecule AHL with a certain concentration can obviously enhance the pathogenic bacteria of the chemical pesticideEC1The bactericidal capacity of the pesticide composition is high, and the micromolecule AHL is used as a novel efficient bactericidal pesticide synergist, so that the bactericidal pesticide composition has high utilization value and development prospect in the aspect of preventing and treating agricultural plant diseases.
In combination with the production and application cost and the sterilization effect after consideration, the concentration range of 40-500 mu M of small molecular AHL is selected as the preferable concentration range for preventing and treating the diseases caused by the Dieckea zeae by mixing with chemical pesticides. Preferably, the concentration of the small molecular compound AHL is 40uM to 500uM, and more preferably 160uM to 500 uM. When mixing, it is further preferable that the concentration of the small molecule compound AHL is 320uM, or 500 uM.
The invention has the following beneficial effects: the invention starts from a new direction of developing the sterilization synergist, utilizes the signal molecule AHL generated by the bacteria to be mixed with the chemical pesticide, and the result shows that the pesticide effect of the chemical bactericide can be obviously improved, the use amount and the pesticide residue of the bactericide are greatly reduced, the cost can be reduced, the natural product of the bacteria is more environment-friendly, the environment can be protected, the problem that the drug resistance of pathogenic bacteria is increasingly severe is effectively relieved, the drug reduction index and the green development of agriculture are promoted to be achieved, and the invention has important significance for solving the problem of the severe drug resistance of the pathogenic bacteria. The invention determines that the applicable concentration of the micromolecule AHL serving as the synergist of the chemical pesticide bactericide for preventing and treating the diseases caused by the Dieckea zeae is 40 uM-500 uM.
Drawings
FIG. 1 shows the bactericidal and synergistic effects of small-molecule AHL mixed thiabendazole copper on Dieckea zeae (10-fold dilution of treated bacterial liquid). Wherein, 1A: WT (without any treatment of material); 1B: phe (treated with Thiodiazole copper alone); 1C: diluting the agricultural thiabendazole copper by 200 times and mixing with 40 mu M AHL; 1D: diluting the agricultural thiabendazole copper by 200 times and mixing with 80 mu M AHL; 1E: diluting the agricultural thiabendazole copper by 200 times and mixing with 160 mu M AHL; 1F: diluting the agricultural thiabendazole copper by 200 times and mixing with 320 mu M AHL; 1G: diluting the agricultural thiabendazole copper by 200 times and mixing with 500 mu M AHL; note: dilution of treatment solution 104And (5) coating the plate. .
FIG. 2 shows the statistical result of the bactericidal effect of 20% agricultural mixed AHL on Dieckea zeae in terms of Phe, P-40-P-500, Phe, P-40-P-500, and Phe.
FIG. 3 is a statistical result of the bactericidal and synergistic effects of small-molecule AHL mixed agricultural thiabendazole copper on Dieckea zeae. Wherein: P-40-P-500 represents that 40 mu M-500 mu M AHL is selected to be mixed with the agricultural thiodiazole copper treatment bacterial liquid; the synergistic ratio of the different concentrations of AHL as the synergist to the mixed agricultural thiediazole copper = CFU for agricultural thiediazole copper alone/(agricultural thiediazole copper + AHL synergist) CFU.
FIG. 40.5% methanol effect on Dieckea zeae 4A: WT (treated with 5ul sterile water alone); 4B (treated with 5ul of methanol alone).
Detailed Description
The present invention will be described in further detail below with reference to examples and test examples, but the present invention is not limited to the following examples and is applicable to various agricultural chemicals and fungicides.
Example 1 preparation of AHL Small molecules
Inoculating overnight cultured Pseudomonas aeruginosa seed solution into YEB culture medium, 28oC. After culturing at 200rpm for 24 hours, the culture supernatant was collected by centrifugation. The obtained supernatant is used for extracting small molecules. Wherein, the YEB culture medium formula is as follows: 0.5 g/LMgSO4•7H2O, 5 g/L Sucrose, 5 g/L Yeast Extract, 10g/L Trypton, 5 g/L NaCl，pH7.0。
The invention adopts the conventional method for separating and extracting the compounds to separate and extract the active substances in the fermentation liquor. After the culture supernatant was collected by centrifugation, ethyl acetate equivalent to the supernatant was added, and the mixture was allowed to stand at 4 ℃ for 2 hours, then subjected to shaking treatment at 20 ℃ for 1 hour, extracted with a separatory funnel, and the upper layer was separated. And carrying out rotary evaporation on the collected upper layer liquid separation by using a rotary evaporator, evaporating the organic solvent to dryness, and eluting by using 2-3mL of methanol. Evaporating methanol solution containing active ingredient to dryness, concentrating, and further purifying by High Performance Liquid Chromatography (HPLC), analyzing, and collecting active substance. The structure identification confirms that the active ingredient is AHL.
Example 2 Sterilization and synergism of micromolecular AHL and Thiobacillus copper blending on Dieckea zeae
The bacterial colony counting statistical method (CFU method) is adopted to determine the bactericidal synergistic effect of the small molecular AHL extracts with different concentrations on the thiediazole copper: preparing and diluting small molecular AHL to a certain concentration with methanol as solvent, collecting overnight culture to OD600=1.0EC1Adding 100mg/ml of Thiobiazole copper diluted 500 times into the bacterial liquid, setting a treatment without adding small-molecule AHL as a positive control, and setting a series of treatments of mixing the small-molecule AHL with concentration gradients (40 mu M, 80 mu M, 160 mu M, 320 mu M and 500 mu M) with the Thiobiazole copper. All processing is at 28oC. After 24 hours of incubation at 200rpm, the cells were diluted 104From this, 200. mu.L of a bacterial suspension was plated (LB medium) and cultured in a 28 ℃ incubator. And observing and counting the result after about 3-4 days.
Counting the plate colony number can obtain: CFU of 1.25X 10 treated with Thiachopper alone4The CFU diluted 500 times with Thiodiazole copper and mixed with 40 μ M AHL was 1.1 × 104The CFU diluted 500 times with Thiodiazole copper and mixed with 80 μ M AHL was 1.0 × 104The CFU of the mixture diluted 500 times with Thiodiazole copper and 160. mu.M AHL was 9.6X 103The CFU of the mixture diluted 500 times with Thiodiazole copper and 320. mu.M AHL is 7.5X 103The CFU of the mixture diluted 500 times with Thiodiazole copper and 500. mu.M AHL is 5.3X 103(see FIGS. 1 and 2). Sterilization synergistic ratio of thiediazole copper by using 40 mu M small-molecule AHLThe value is 1.06 times, the ratio of the bactericidal synergy of 80 μ M of the small-molecule AHL to the copper thienyls is 1.14 times, the ratio of the bactericidal synergy of 160 μ M of the small-molecule AHL to the copper thienyls is 1.27 times, the ratio of the bactericidal synergy of 320 μ M of the small-molecule AHL to the copper thienyls is 2.90 times, and the ratio of the bactericidal synergy of 500 μ M of the small-molecule AHL to the copper thienyls is 2.77 times (see fig. 3). 0.5% methanol had no significant effect on Dieckea zeae (see FIG. 4).
Claims (9)
1. The application of the small molecular compound AHL as a synergist of a chemical agent for preventing and treating plant diseases caused by the Dieckea zeae, wherein the chemical agent for preventing and treating the plant diseases caused by the Dieckea zeae is Thiodiazole; the small molecule compound AHL has the following structure:
2. use according to claim 1, characterized in that: the plant disease is soft rot.
3. Use according to claim 1, characterized in that: the plant disease is rice bacterial basal rot or banana bacterial soft rot.
4. Use according to claim 1, characterized in that: the small molecular compound AHL is extracted from pseudomonas aeruginosa fermentation liquor or artificially and chemically synthesized.
5. The use according to claim 1, wherein the chemical agent is 20% copper thielavone and is used in a 500-fold dilution.
6. The use according to any one of claims 1 to 5, characterized in that the small molecular compound AHL is mixed with a chemical agent for controlling plant diseases caused by the Dieckea zeae to increase the control effect.
7. The use according to claim 6, wherein the concentration of the small molecule compound AHL is 40uM to 500uM when the small molecule compound AHL is mixed.
8. The use according to claim 7, wherein the concentration of the small molecule compound AHL is 160 to 500uM when compounded.
9. The use according to claim 8, wherein the concentration of the small molecule compound AHL is 320uM, or 500uM, when compounded.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110105597A1 (en) * | 2002-03-08 | 2011-05-05 | Aldo Ammendola | Modulation of pathogenicity |
| CN103781353A (en) * | 2011-09-09 | 2014-05-07 | 拜耳知识产权有限责任公司 | Acyl-homoserine lactone derivatives for improving plant yield |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110105597A1 (en) * | 2002-03-08 | 2011-05-05 | Aldo Ammendola | Modulation of pathogenicity |
| CN103781353A (en) * | 2011-09-09 | 2014-05-07 | 拜耳知识产权有限责任公司 | Acyl-homoserine lactone derivatives for improving plant yield |
Non-Patent Citations (6)
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| Characterization of pectolytic erwinias as highly sophisticated pathogens of plants;Solke H. De Boer;《European Journal of Plant Pathology》;20031231;第109卷;893–899 * |
| Denis Faure •Yves Dessaux.Quorum sensing as a target for developing control strategies for the plant pathogen Pectobacterium.《Eur J Plant Pathol》.2007,第119卷353–365. * |
| New Synthetic Analogues of N-Acyl Homoserine Lactones as Agonists or Antagonists of Transcriptional Regulators Involved in Bacterial Quorum Sensing;Sylvie Reverchon et.al.;《Bioorganic & Medicinal Chemistry Letters》;20021231;第12卷;1153–1157 * |
| Quorum Sensing Inhibition and Structure–Activity Relationships of β-Keto Esters;Stephanie Forschner-Dancause et.al.;《Molecules》;20160725;第21卷;971-979 * |
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