CN108077257B - Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control - Google Patents

Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control Download PDF

Info

Publication number
CN108077257B
CN108077257B CN201711153968.8A CN201711153968A CN108077257B CN 108077257 B CN108077257 B CN 108077257B CN 201711153968 A CN201711153968 A CN 201711153968A CN 108077257 B CN108077257 B CN 108077257B
Authority
CN
China
Prior art keywords
dsf
streptomycin sulfate
shenqinmycin
cfu
citrus canker
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
Application number
CN201711153968.8A
Other languages
Chinese (zh)
Other versions
CN108077257A (en
Inventor
常长青
刘琼光
张炼辉
邓嘉茹
吴熔熔
仇善旭
陈红彩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN201711153968.8A priority Critical patent/CN108077257B/en
Publication of CN108077257A publication Critical patent/CN108077257A/en
Application granted granted Critical
Publication of CN108077257B publication Critical patent/CN108077257B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/06—Unsaturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • 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
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/60—1,4-Diazines; Hydrogenated 1,4-diazines
    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
    • A01N47/18—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof containing a —O—CO—N< group, or a thio analogue thereof, directly attached to a heterocyclic or cycloaliphatic ring
    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids

Abstract

The invention discloses a citrus canker (A)Xanthomonas axonopodis pv.citri) The micromolecule DSF is used as a synergist of antibiotic bactericides, and specifically comprises the following components: the addition of small molecular compounds to mainstream antibiotic bactericides (such as agricultural streptomycin sulfate, zhongshengmycin, shenqinmycin and the like) can significantly increase the bactericidal effect of antibiotics. The DSF is derived from citrus cankerXacExtracting in fermentation liquor or artificially chemically synthesizing. The synergist can obviously improve the bactericidal effect of the antibiotic bactericide, greatly reduce the use amount of chemical pesticides, weaken the drug resistance of pathogenic bacteria and finally promote the harmonious development of the planting industry and the environment.

Description

Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control
Technical Field
The invention relates to the field of plant protection by biological control technology, in particular to citrus cankerXanthomonas axonopodis pv.citri（Xac) The produced micromolecular DSF substances are green and safeThe novel synergist can improve the bactericidal effect of antibiotic bactericides such as streptomycin sulfate, shenqinmycin and zhongshengmycin, and reduce the application amount of chemical pesticides in disease control.
Background
In the comprehensive prevention and control of plant diseases, chemical pesticide control is widely favored due to the characteristics of quick response, remarkable effect and the like, and China is one of the most serious countries in the world abusing chemical agents. With the concern and importance of people on personal health, food safety and ecological environment, the reduction of chemical agents, especially the dosage of traditional high-toxicity and high-residue chemical pesticides, is imperative in agricultural production. Research and develop and popularize novel pesticides with low toxicity, low residue, good environmental compatibility and remarkable prevention and treatment effect; the research and development of novel pesticide synergist can increase pesticide effect and reduce pesticide consumption, and has urgent and bright prospect. The traditional synergist improves the pesticide effect of chemical pesticides by improving the wetting, spreading, dispersing, detention, permeability and the like of the pesticides, but has the defects of difficult synthesis, environmental pollution and the like. Starting from the perspective of pathogenic bacteria, the bactericide effect of the pesticide is improved by changing the life habits of pathogenic microorganisms and inhibiting and weakening the drug resistance of pathogenic bacteria, the usage amount of the pesticide is reduced, the cost is reduced, and the protection of the ecological environment is the development direction of a novel synergist in the future, so that the pesticide synergist not only can prolong the life cycle of pesticide varieties, but also is popular due to the green safety characteristic of the pesticide varieties.
The earlier stage research of the invention finds the pathogen of citrus cankerXacProducing novel small molecules with different structures. Such as Wang et al (Wang L, He Y, Gao Y, et al.. A bacterial cell-cell communication signal with cross-kingdom structural analogues[J]molecular Microbiology, 2004,51(3):903 one 912.), Ellen et al (Beaulieu E D, Ionescu M, Chatterjee S, et al, Characterisation of a Difusable signalling Factor from Xylella Fastica [ J ]]Mbio, 2013, 4(1): 00539-12) disclose DSF,XfThe structure of the DSF is as follows:
Figure 507573DEST_PATH_IMAGE001
Figure 309307DEST_PATH_IMAGE002
。
however, no report on the aspect of improving the bactericidal effect of medicaments by researching micromolecular DSF substances exists at present.
Disclosure of Invention
The research of the inventor finds that pathogenic bacteria are added into agricultural streptomycin sulfate serving as the citrus canker prevention and control agent indoors or in the fieldXacThe generated micromolecular DSF substances can obviously improve the sterilization effect of the medicament, thereby reducing the using amount of the medicament and the reagent. The small molecule DSF substances refer to DSF and/orXfDSF。
The research on the mixed use of micromolecular DSF substances as a synergist and antibiotic bactericides can obviously improve the pesticide effect of the bactericides, reduce the use amount of chemical agents, reduce the cost and have important significance on solving the increasingly prominent resistance problem of pathogenic bacteria to the bactericides. The use of small molecular DSF substances as a synergist and pesticides for preventing and treating plant diseases is not reported.
The invention aims to provide a new application of micromolecular DSF substances.
The purpose of the invention is realized by the following technical scheme:
provides the application of micromolecular DSF substances as a synergist and an antibiotic bactericide in preventing and treating citrus canker.
The micromolecular DSF substances are derived from citrus canker pathogenic bacteriaXanthomonas axonopodis pv.citriExtracting in fermentation culture solution or artificially chemically synthesizing.
The extraction method of the micromolecular DSF substances is to collect the citrus canker pathogen by centrifugationXacAdding ethyl acetate into the culture supernatant overnight, extracting, separating and purifying by High Performance Liquid Chromatography (HPLC), collecting active substances, and identifying by structureXacThe generated small molecule DSF,XfDSF。
Separating, extracting and preparing micromolecular DSF substances with different concentrations and antibioticsBactericide (agricultural streptomycin sulfate, shenqinmycin and zhongshengmycin) mixed treatment on pathogenic bacteriaXacCounting the growth of colony, comparing the mixed small molecules with different concentrations with antibiotic bactericide to treat pathogenic bacteria on indoor and potted citrusXacThe sterilization effect of (1). Test results show that micromolecular DSF with certain concentration can obviously improve the effect of the antibiotic bactericide on pathogenic bacteriaXacThe bactericidal effect of the composition is developed into a novel efficient bactericidal synergist which has bright prospect in preventing and treating plant diseases.
Based on the control of the later production and application cost, the invention provides that the micromolecule DSF substances and the antibiotic bactericide are mixed and prepared to be applied to the prevention and treatment of citrus canker in the concentration range of 0.01 mu M-1 mM, and the more preferable concentration range is 10 mu M-500 mu M.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention starts from pathogenic bacteria, can remarkably improve the drug effect by mixing natural unsaturated fatty acid micromolecule DSF substances generated by bacteria with antibiotic bactericides (agricultural streptomycin sulfate, shenqinmycin and zhongshengmycin), provides a new thought for researching and developing a novel green and environment-friendly bactericidal synergist by reducing the dosage of the bactericides and has important significance for solving the increasingly prominent drug resistance problem of the pathogenic bacteria.
2) The technical proposal that the citrus canker germ DSF substance is used as a synergist for preventing and controlling citrus bacterial plant diseases is determined.
Drawings
FIG. 1 Small moleculeXfThe DSF mixed with agricultural streptomycin sulfate has a sterilization synergistic effect on the citrus canker bacteria (10 times of the treated bacterial liquid). Note: 1A: WT +5% CH3OH; 1B: str (treatment with streptomycin sulphate alone for agricultural use); 1C: agricultural streptomycin sulfate 500 times and 20 mu MXfMixing DSF; 1D: agricultural streptomycin sulfate 500 times and 40 mu MXfMixing DSF; 1E: agricultural streptomycin sulfate 500 times and 60 mu MXfMixing DSF;
FIG. 2 Small moleculesXfMethod for crushing citrus by DSF mixed agricultural streptomycin sulfateAnd (5) statistics of sterilization and synergism of the ulcer fungi. Wherein: note: str represents the 500-fold treatment of the inoculum with streptomycin sulphate alone (as a positive control); s-20 to S-100 is 20 to 100 μ MXfMixing DSF with agricultural streptomycin sulfate treatment bacterial liquid; signals of different concentrationsXfThe synergy ratio of DSF as synergist to streptomycin = streptomycin used alone CFU/(streptomycin +)XfDSF synergist) CFU
FIG. 3 shows that the bactericidal synergistic effect of small molecular DSF mixed with agricultural streptomycin sulfate on citrus canker bacteria (10 times of the treated bacteria liquid dilution). Note: 3A: WT +5% CH3OH; 3B: str (treatment with streptomycin sulphate alone for agricultural use); 3C: 500 times of agricultural streptomycin sulfate is mixed with 100 mu M DSF; 3D: 500 times of agricultural streptomycin sulfate is mixed with 200 mu M DSF; 3E: 500 times of agricultural streptomycin sulfate is mixed with 300 mu M DSF;
FIG. 4 is a statistical result of the bactericidal synergistic effect of micromolecule DSF mixed agricultural streptomycin sulfate on citrus canker. Wherein: note: str represents the 500-fold treatment of the inoculum with streptomycin sulphate alone (as a positive control); S-50-S-500 represents that 50 mu M-500 mu M DSF is selected to be mixed with agricultural streptomycin sulfate treated bacteria liquid; the synergy ratio of different concentration signals DSF as potentiators to streptomycin mixed with streptomycin = streptomycin CFU/(streptomycin + DSF potentiator) CFU alone.
FIG. 5 Small moleculeXfSterilization and synergism of DSF mixed with shenqinmycin on citrus canker (bacterial liquid treating diluent 10)-3Multiple). Wherein: note: 5A: WT +5% CH3OH; 5B: shen (shenqinmycin alone); 5C: 500 times and 20 mu M of shenqinmycinXfMixing DSF; 5D: 500 times and 40 mu M of shenqinmycinXfMixing DSF; 5E: 500 times and 60 mu M of shenqinmycinXfMixing DSF;
FIG. 6 shows the bactericidal and synergistic effects of small molecular DSF mixed shenqinmycin on citrus canker (10 percent of treated bacterial liquid)-3Multiple). Wherein: note: 6A: WT +5% CH3OH; 6B: shen (shenqinmycin alone); 6C: 500 times and 50 mu M of shenqinmycinXfMixing DSF; 6D: 500 times and 100 mu M of shenqinmycinXfMixing DSF; 6E: 500 times and 200 mu M of shenqinmycinXfMixing DSF; 6F: 500 times and 300 mu of shenqinmycinM XfMixing DSF;
FIG. 7 Small moleculesXfSterilization and synergism of DSF mixed zhongshengmycin on citrus canker (treated bacterial liquid diluent 10)-3Multiple). Wherein: note: 7A: WT +5% CH3OH; 7B: z (treated with zhongshengmycin alone); 7C: zhongshengmycin 5000 times and 20 mu MXfMixing DSF; 7D: zhongshengmycin 5000 times and 40 mu MXfMixing DSF; 7E: zhongshengmycin 5000 times and 60 mu MXfMixing DSF;
FIG. 8 shows the bactericidal and synergistic effect of mixed zhongshengmycin with small molecular DSF on citrus canker (10 percent of treated bacterial liquid)-3Multiple). Wherein: note: 8A: WT +5% CH3OH; 8B: z (treated with zhongshengmycin alone); 8C: 5000 times of zhongshengmycin is mixed with 50 mu M DSF; 8D: 5000 times of zhongshengmycin is mixed with 100 mu M DSF; 8E: mixing the zhongshengmycin 5000 times and 200 mu M DSF;
FIG. 9 Small moleculesXfAnd (3) counting the potted plant inoculation result of the sterilization and synergism effect of the DSF mixed agricultural streptomycin sulfate on the citrus canker. Wherein: note: str is expressed as the inoculated leaves treated with 500-fold solution of agricultural streptomycin sulfate alone (as a positive control); s-60 denotes 60. mu.MXfMixing DSF with agricultural streptomycin sulfate 500 times to treat the inoculated leaves; s-80 denotes 80. mu.MXfMixing DSF with agricultural streptomycin sulfate 500 times to treat the inoculated leaves; s-100 denotes 100. mu.MXfMixing DSF with agricultural streptomycin sulfate 500 times to treat the inoculated leaves;
FIG. 10 is a statistical result of potting inoculation of the bactericidal synergistic effect of small molecular DSF mixed with agricultural streptomycin sulfate on citrus canker. Wherein: note: str is expressed as the inoculated leaves treated with 500 times of streptomycin sulfate solution alone (as a positive control); s-300 represents the inoculated leaves were treated with 500 times of 300. mu.M DSF mixed with streptomycin sulphate for agricultural use; s-400 represents that the inoculated leaves are treated by 500 times of 400 MuM DSF mixed agricultural streptomycin sulfate; s-500 represents 500-fold treatment of inoculated leaves with 500. mu.M DSF mixed streptomycin;
FIG. 11 shows that the micromolecule DSF is mixed with agricultural streptomycin sulfate as a synergist to kill and synergize pathogenic bacteria of rice basal rot. Wherein: note: FIG. 11A shows the addition of a 0.5% methanol control to the culture medium; 11B is represented byTreating the bacterial liquid by using 500 times of agricultural streptomycin sulfate solution; 11C is 80. mu.MXfDSF is mixed with agricultural streptomycin sulfate treatment bacterial liquid. FIG. 11D shows the addition of a 0.5% methanol control to the medium; 11E represents that the agricultural streptomycin sulfate 500 times solution is used for treating the bacterial liquid independently; 11F is 200 μ M DSF mixed with agricultural streptomycin sulfate treated bacterial liquid.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.
The mother liquor concentration of 72% streptomycin sulfate for agricultural use employed in examples 1 to 10 was 100mg/mL, and the working concentration was 500 times the dilution; the concentration of the mother liquor of 3 percent Zhongshengmycin is 10mg/mL, and the working concentration is 5000 times of the diluent; the concentration of the 1% shenqinmycin mother liquor is 10mg/mL, and the working concentration is 500 times of the diluent. The test concentration of small molecule DSF was 100 mM. The embodiment of the invention shows that the micromolecule DSF with a certain concentration range and the antibiotic bactericide are mixed to be used as a biological source synergist to have certain bactericidal synergism on the prevention and treatment of the citrus canker, and the combination of a part of antibiotic bactericide and micromolecules serving as the synergist can achieve the synergism according to the technical characteristics of the invention.
Example 1 DSF and derivatives thereofXfPreparation of DSF
Will be cultured overnightXacInoculating seed solution into YEB culture medium, culturing at 28 deg.C and 200rpm for 24 hr, centrifuging, and collecting Citrus canker germXacAnd (4) supernatant of the culture solution. The obtained supernatant is used for extracting small molecules.
The invention adopts the conventional method for separating and extracting compounds to separate and extract active substances in fermentation liquor (see two prior art documents mentioned in the background of the specification). Centrifugally collecting citrus canker germsXacAdding ethyl acetate with the same volume as the supernatant after the culture solution is supernatant, standing at 4 ℃ for 2h, treating at 20 ℃ for 1h by a shaking table, extracting by a separating funnel, and collecting the upper layer of separating liquid. And then suspending and evaporating the collected upper layer of separated liquid by using a rotary evaporator, evaporating the organic solvent to dryness, and eluting by using 2-3mL of methanol. Will contain active ingredientsThe methanol solution was evaporated to dryness and concentrated, and the active substance was further purified, analyzed and collected by High Performance Liquid Chromatography (HPLC). Performing structural identification on the active ingredient, and confirming that the active ingredient is DSF and derivatives thereof respectivelyXfDSF。
Example 2XfThe DSF and 72% agricultural streptomycin sulfate are mixed to be matched with citrus canker pathogenXacThe sterilization and synergism of
Dissolving the extract by using methanol as a solvent, mixing the dissolved extract with a 100mg/mL agricultural streptomycin sulfate reagent to treat the citrus canker bacterial liquid with a certain OD value, and measuring the sterilization synergistic effect of the extract with different concentrations on the agricultural streptomycin sulfate by using the total bacterial Community (CFU) in unit volume. The specific operation is as follows:
adopting a statistical CFU method: using methanol as solventXfDSF is prepared into a test medicament with a certain concentration, and the test medicament is accurately addedXfMixing DSF solution with 500 times of agricultural streptomycin sulfate reagent to culture overnight to OD600=1.0XacIn the bacterial liquid, different concentrations are addedXfDSF, 500 times of 72% agricultural streptomycin sulfate reagent and bacterial liquid are treated simultaneously, 500 times of 72% agricultural streptomycin sulfate treated bacterial liquid is used as positive control singly, and the positive control is placed in 28oC. After 24 hours of culture at 200rpm, the treated cells were diluted in gradient and plated with 200. mu.L of the treated cells (LB medium). Duplicate plates were plated at 3 concentrations each. After several days in the 28 ℃ incubator, the experimental results were observed and counted: with reference to FIG. 1, plate colony counts were calculated as follows: CFU for the untreated control CK 1A was 5X 1081B CFU treated with streptomycin sulfate alone for agricultural use 15X 105500 times and 20 mu M of 1C agricultural streptomycin sulfateXfThe CFU of the DSF mixing treatment is 4 multiplied by 105500 times and 40 mu M of 1D agricultural streptomycin sulfateXfThe CFU of the DSF compounding treatment is 2.5 multiplied by 105500 times and 60 mu M of 1E agricultural streptomycin sulfateXfThe CFU of the DSF mixing treatment is 1 multiplied by 104. In conjunction with fig. 2, it follows: s-20 is expressed as a small molecule at 20. mu.MXfThe sterilization synergistic ratio of the DSF to the agricultural streptomycin sulfate is 3 times, and S-40 is expressed as 40 mu M micromoleculeXfThe sterilization synergistic ratio of the DSF to the agricultural streptomycin sulfate is 6 times, and S-60 is expressed as 60 mu M micromoleculeXfMethod for preparing agricultural streptomycin sulfate by DSFThe sterilization synergistic ratio is 150 times, S-80 is expressed as small molecule of 80 mu MXfThe sterilization synergistic ratio of the DSF to the agricultural streptomycin sulfate is 60 times, and S-100 is expressed as 100 mu M micromoleculeXfThe sterilization synergistic ratio of the DSF to the agricultural streptomycin sulfate is 300 times.
Example 3 DSF in combination with 72% agricultural streptomycin sulfate is used to treat citrus cankerXacThe sterilization and synergism of
According to an experimental method similar to that of example 2, the bactericidal synergistic effect of DSF and 72% agricultural streptomycin sulfate on citrus canker pathogenic bacteria Xac is studied. As can be seen from FIG. 3, the CFU of the untreated control CK of 3A is 4X 1083B CFU of 10X 10 treated with streptomycin sulphate for agricultural use alone5The CFU of the mixed treatment of 500 times of the 3C agricultural streptomycin sulfate and 100 MuM DSF is 1 multiplied by 105The CFU of the 3D agricultural streptomycin sulfate mixed with 200 mu M DSF is 4 multiplied by 104The CFU of the mixed treatment of 500 times of the 3E agricultural streptomycin sulfate and 300 mu M DSF is 2 multiplied by 104. In conjunction with fig. 4, it follows: s-50 represents that the ratio of the bactericidal effect of 50 mu M micromolecular DSF to the agricultural streptomycin sulfate is 1.5 times, S-100 represents that the ratio of the bactericidal effect of 100 mu M micromolecular DSF to the agricultural streptomycin sulfate is 2 times, S-200 represents that the ratio of the bactericidal effect of 200 mu M micromolecular DSF to the agricultural streptomycin sulfate is 3 times, S-300 represents that the ratio of the bactericidal effect of 300 mu M micromolecular DSF to the agricultural streptomycin sulfate is 50 times, and S-400 represents that the ratio of the bactericidal effect of 400 mu M micromolecular DSF to the agricultural streptomycin sulfate is 150 times. S-500 represents that the bactericidal synergistic ratio of 500 mu M of micromolecular DSF to agricultural streptomycin sulfate is 140 times.
Example 4XfMixing DSF and 1% shenqinmycin for citrus cankerXacThe sterilization and synergism of
The extract is dissolved by using methanol as a solvent, the extract is mixed with a shenqinmycin reagent of 10mg/mL to treat the citrus canker pathogen liquid with a certain OD value, and the bacterial colony Count (CFU) in unit volume is used for measuring the sterilization synergistic effect of the extract with different concentrations on the shenqinmycin. The specific operation is as follows:
adopting a statistical CFU method: using methanol as solventXfTest with DSF configured to a concentrationThe medicament is added accurately in a certain amountXfDSF solution mixed 500 times shenqinmycin reagent to overnight culture to OD600=1.0XacIn the bacterial liquid, different concentrations are addedXfDSF, 500-time shenqinmycin and bacterial liquid are treated simultaneously, 500-time shenqinmycin treated bacterial liquid is used as positive control singly and is placed in 28oC. After 24 hours of culture at 200rpm, the treated cells were diluted in gradient and plated with 200. mu.L of the treated cells (LB medium). Duplicate plates were plated at 3 concentrations each. After several days in the 28 ℃ incubator, the plate colony count is counted in conjunction with FIG. 5 to obtain: CFU for the untreated control CK 5A 5X 1085B CFU treated with shenqinmycin alone 5X 1065C shenqinmycin 500 times and 20 mu MXfThe CFU of the DSF compounding treatment is 2.5 multiplied by 1065D agricultural streptomycin sulfate 500 times and 40 mu MXfThe CFU of the DSF blending treatment is 106 5E shenqinmycin 500 times and 60 mu MXfThe CFU of the DSF mixing treatment is 4 multiplied by 105 5F shenqinmycin 500 times and 80 mu MXfThe CFU of the DSF compounding treatment is 1.5 multiplied by 105。
Example 5 compounding of DSF with 1% shenqinmycin against Leptosphaeria citricolaXacThe sterilization and synergism of
According to an experimental method similar to that of example 4, the bactericidal synergistic effect of DSF and 1% shenqinmycin on citrus canker pathogenic bacterium Xac is studied. With reference to FIG. 6, plate colonies were counted: CFU for 6A untreated control CK was 5X 1086B CFU treated with shenqinmycin alone was 5X 106CFU of the mixture of 500 times of 6C shenqinmycin and 50 MuM DSF is 2.5 multiplied by 106The CFU of the 6D agricultural streptomycin sulfate mixed with 100 MuM DSF is 2 multiplied by 106CFU of 6E shenqinmycin mixed with 200 μ M DSF is 4 × 105CFU of the mixture of 500 times of 6F shenqinmycin and 300 mu M DSF is 3 multiplied by 104。
Example 6XfThe mixture of DSF and 3% Zhongshengmycin can be used for treating citrus cankerXacThe sterilization and synergism of
The extract is dissolved by using methanol as a solvent, the extract is mixed with 10mg/mL zhongshengmycin reagent to treat the citrus canker bacterial liquid with a certain OD value, and the total bacterial colony Count (CFU) in unit volume is used for measuring the bactericidal synergistic effect of the extract with different concentrations on the zhongshengmycin. The specific operation is as follows:
adopting a statistical CFU method: using methanol as solventXfDSF is prepared into a test medicament with a certain concentration, and a certain amount of DSF is accurately addedXfDSF solution was mixed with 500-fold Zhongshengmycin reagent to OD overnight600=1.0XacIn the bacterial liquid, different concentrations are addedXfDSF, 500 times zhongshengmycin and bacterial liquid are treated simultaneously, 500 times zhongshengmycin treated bacterial liquid is used alone as positive control and is placed in 28oC. After 24 hours of culture at 200rpm, the treated cells were diluted in gradient and plated with 200. mu.L of the treated cells (LB medium). Duplicate plates were plated at 3 concentrations each. After several days in the 28 ℃ incubator, the plate colony count is counted in conjunction with FIG. 7 to obtain: CFU for 7A untreated control CK was 5X 1087B CFU treated with Zhongshengmycin alone was 1085000 times and 20 mu M of zhongshengmycin in 7CXfThe CFU of the DSF blending treatment is 1075000 times and 40 mu M Zhongshengmycin for 7DXfThe CFU of the DSF compounding treatment is 1.2 multiplied by 1075000 times and 60. mu.M Zhongshengmycin, 7EXfThe CFU of the DSF compounding treatment is 1.5 multiplied by 107。
Example 7 DSF in combination with 3% ZhongshengmycinXacThe sterilization and synergism of
The bactericidal synergistic effect of DSF in combination with 3% zhongshengmycin on Leptospira citrea Xac was studied in a manner similar to the experimental procedure of example 6. With reference to FIG. 8, plate colony counts were counted as: CFU of 8A untreated control CK was 5X 1088B CFU treated 5000-fold with Zhongshengmycin alone was 5X 106And the CFU of the 8C mixed treatment of 5000 times of zhongshengmycin and 50 mu M DSF is 106The CFU of 8D mixed treatment of 5000 times of zhongshengmycin and 100 MuM DSF is 5 multiplied by 1058E, the CFU of the mixture treatment of 5000 times of zhongshengmycin and 200 mu M DSF is 105。
EXAMPLE 8XfGreenhouse potting test for preventing and controlling citrus canker disease germs by mixing DSF and agricultural streptomycin sulfate as synergist
Citrus canker (A)Xanthomonas axonopodis pv.citri) Preparation of a bacterial liquid suspension: will be on LB mediumAfter streaking for 2 days, single colony was inoculated into LB liquid medium and OD was cultured overnight in a 28 ℃ incubator600=0.5, ready for inoculation.
The outdoor potting experiment was carried out in a plastic greenhouse. The method comprises purchasing a potted plant of a disease-susceptible variety of the four-season mandarin orange planted for more than 2 years as a test material, selecting four-season mandarin orange tender leaves with the same size, similar color and same growth as an experimental inoculation object, sterilizing the surfaces of the tender leaves with 75% alcohol before inoculation, piercing the leaves from the back of the leaves with a syringe needle, uniformly piercing 8 perforations at two sides of the far axis of the leaves, and inoculating 5 mu L of OD (origin-destination) above the perforations600And (4) inoculating the bacterial liquid suspension of which the concentration is 0.5, and then culturing the bacterial liquid suspension under the conditions of room temperature and natural illumination, and performing conventional water and fertilizer management.
Respectively spraying the tender leaves of the four seasons orange after being inoculated with the pathogenic bacteria for 3 days with tap water with different concentrationsXfDSF takes a test reagent mixed with 500 times of agricultural streptomycin sulfate and a treatment reagent taking 500 times of agricultural streptomycin sulfate as positive control, LB culture medium as negative control, 1mL of reagent is sprayed on each tender leaf, and each treatment is repeated for 3 times.
Agricultural streptomycin sulfate medicament blending with different concentrationsXfAfter the citrus fruit leaves inoculated with pathogenic bacteria are treated by the DSF for three days, sampling is carried out, areas with scabs of each treated leaf are cut into a sterilized mortar, a small amount of quartz sand and 2mL of LB culture medium are added, the mixture is ground into a homogenate state to be arranged in a 5mL centrifuge tube, 3mL of LB culture medium is added, the mixture is shaken up and filtered by a sterilized gauze. Suspending the obtained filtrate with 10000g/2 min and 1mL of culture medium, repeating the steps twice, suspending with 200 muL of LB culture medium, diluting different concentration gradients of 200 muL coated plates, observing the growth density of colony plates, counting, and obtaining the result by combining the results of FIG. 9: : s-60 is expressed as a small molecule at 60. mu.MXfThe bactericidal synergy ratio of the DSF to the agricultural streptomycin sulfate is 1.5 times, and S-80 is expressed as 80 mu M micromoleculeXfThe sterilization synergistic ratio of the DSF to the agricultural streptomycin sulfate is 2 times, and S-100 is expressed as 100 mu M micromoleculeXfThe bactericidal synergistic ratio of the DSF to the agricultural streptomycin sulfate is 10 times.
Greenhouse potting test for preventing and controlling citrus canker pathogen by using 9 DSF and agricultural streptomycin sulfate mixed as synergist
According to an experimental method similar to that of example 8, a greenhouse potting experiment for preventing and controlling citrus canker pathogenic bacteria by mixing DSF and agricultural streptomycin sulfate as a synergist is researched. The results in conjunction with fig. 10 yield: : s-300 shows that the ratio of sterilization and synergism of 300 mu M micromolecular DSF to agricultural streptomycin sulfate is 2.5 times, S-400 shows that the ratio of sterilization and synergism of 400 mu M micromolecular DSF to agricultural streptomycin sulfate is 15 times, and S-500 shows that the ratio of sterilization and synergism of 500 mu M micromolecular DSF to agricultural streptomycin sulfate is 7.5 times.
The 10 DSF and the derivatives thereof are mixed with agricultural streptomycin sulfate to be used as a synergist for treating rice basal rot pathogenic bacteriaEC1The sterilization and synergism of
Methanol is used as a solvent to dissolve the extract, the extract is mixed with a 100mg/mL agricultural streptomycin sulfate reagent to treat the rice basal rot pathogenic bacteria liquid with a certain OD value, and the total bacterial community number (CFU) in unit volume is used to determine the sterilization synergistic effect of the extract with different concentrations on the agricultural streptomycin sulfate. The specific operation is as follows:
adopting a statistical CFU method: using methanol as solvent to react DSF andXfthe DSF is respectively prepared into a test medicament with a certain concentration, and a certain amount of DSF or DSFXfMixing DSF solution with 500 times of agricultural streptomycin sulfate to OD after overnight culture600=1.0EC1Adding DSF orXfDSF, 500-fold agricultural streptomycin sulfate and bacterial liquid are treated simultaneously, 500-fold agricultural streptomycin sulfate treated bacterial liquid is used alone as a positive control, the bacterial liquid is cultured at 28 ℃ and 200rpm for 24 hours, then the treated bacterial liquid is diluted in a gradient mode, and 200 mu L of bacterial liquid treated liquid is used for coating a plate (a culture medium is LB). Duplicate plates were plated at 3 concentrations each. After several days in the 28 ℃ incubator, the experimental results were observed and counted: CFU of 11A untreated control CK was 5X 10811B treatment with streptomycin sulphate for agricultural use aloneEC1CFU of (1) is 2.5X 104Agricultural 11C streptomycin sulfate 500 times and 20 mu MXfThe CFU of the DSF mixing treatment is 4 multiplied by 10311D untreated control CK had a CFU of 5X 10811E treatment with streptomycin sulphate for agricultural use aloneEC1CFU of (1) is 2.5X 10411F agricultural streptomycin sulfate 500CFU of 1.5X 10 times the weight of the blend treated with 300. mu.M DSF3。

Claims (6)

1. The antibiotic bactericide compounding preparation is characterized in that antibiotic bactericide of shenqinmycin is taken as an active ingredient, and orange canker pathogenic bacteria (A), (B), (C) and (C) are further addedXanthomonas axonopodis pv. citri) Small molecule DSF orXfDSF, wherein the small molecule DSF orXfThe concentration of DSF is 0.01. mu.M-1 mM.
2. The compounded formulation according to claim 1, wherein for shenqinmycin, the stock solution concentration using 1% shenqinmycin is 10mg/mL, and the working concentration is 500 times the dilution.
3. The compounded formulation of claim 1 or 2, wherein the citrus canker pathogen is (a: (b))Xanthomonas axonopodis pv. citri) The preparation method of the micromolecular DSF substances comprises the following steps:
(1) inoculating the seed liquid of the overnight cultured bacteria into a YEB culture medium, and performing shaking culture;
(2) collecting the culture supernatant by centrifugation;
(3) adding ethyl acetate with the same volume as the supernatant, standing, and treating in a normal temperature shaking table;
(4) extracting with a separating funnel, and collecting upper layer liquid;
(5) suspending and evaporating the collected upper layer of separated liquid by using a rotary evaporator, evaporating the organic solvent to dryness, and eluting by using methanol;
(6) the methanol solution containing the active ingredient is evaporated to dryness and concentrated, and then the active substance is further purified, analyzed and collected.
4. The compounded formulation according to claim 3, wherein the cultivation in step (1) is 28oC. The culture was carried out at 200rpm for 24 hours.
5. The compounded formulation according to claim 3, wherein the standing in step (3) is standing at 4 ℃.
6. The compounded formulation according to claim 3, wherein the shaking table treatment time in step (3) is 2 h.
CN201711153968.8A 2017-11-19 2017-11-19 Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control Active CN108077257B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711153968.8A CN108077257B (en) 2017-11-19 2017-11-19 Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711153968.8A CN108077257B (en) 2017-11-19 2017-11-19 Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control

Publications (2)

Publication Number Publication Date
CN108077257A CN108077257A (en) 2018-05-29
CN108077257B true CN108077257B (en) 2020-12-29

Family

ID=62172648

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711153968.8A Active CN108077257B (en) 2017-11-19 2017-11-19 Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control

Country Status (1)

Country Link
CN (1) CN108077257B (en)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Diffusible signal factor (DSF) quorum sensing signal and structurally related molecules enhance the antimicrobial efficacy of antibiotics against some bacterial pathogens;Yinyue Deng 等;《BMC Microbiology》;20141231;第1-9页 *
新的群体感应信号分子-DSF;田静等;《中国化学会第七届有机化学学术会议分组邀请报告摘要》;20111112;第71页 *
柑橘溃疡病防控的新型杀菌增效剂研究;邓嘉茹等;《中国植物病理学会2017年学生年会论文集》;20170725;第412页 *

Also Published As

Publication number Publication date
CN108077257A (en) 2018-05-29

Similar Documents

Publication Publication Date Title
CN105475303B (en) A kind of composition pesticide containing kasugarnycin and gold alloy hydride
CN102177921B (en) Compound biological control agent as well as preparation method and application thereof
CN104604947A (en) Rice planthopper killing metarhizium anisopliae preparation and application thereof
CN104255757A (en) Compound bactericide
CN108041038B (en) Application of micromolecules generated by citrus canker pathogen as copper preparation bactericide synergist in disease control
CN108077257B (en) Application of micromolecules generated by citrus canker pathogenic bacteria as antibiotic bactericide synergist in disease control
CN101619293B (en) Streptomyces vinaceusdrappus, filtering method and application
CN108094428B (en) Application of citrus canker pathogen-generated micromolecules as thiazole bactericide synergist in disease control
CN105002120A (en) Bacillus mycoides strain and application thereof
CN103314986B (en) Cycloxaprid and spirotetramat-containing insecticidal composition
CN108576018B (en) Application of AHL molecule as chemical pesticide sterilization synergist in disease control caused by Dieckea zeae
CN108513983B (en) Application of AHL molecule as chemical pesticide sterilization synergist in prevention and treatment of citrus canker
CN108651459A (en) A method of the prevention water rice brown spot based on capsaicine
CN108513984B (en) Application of AHL molecule as chemical pesticide sterilization synergist in prevention and treatment of black rot caused by xanthomonas
CN113519541B (en) Application of euphorbia factor L3 in preparation of pesticide preparation for preventing and treating plant oomycetes diseases
CN111944710A (en) Shewanella and application thereof in improving stress resistance of plants
CN111937895B (en) Application of pyraclostrobin and composition containing pyraclostrobin in prevention and treatment of peach branch blight
CN103548832A (en) Acaricidal composition containing pyriminostrobin and cyflumetofen
CN112075449B (en) Composite antibacterial agent for preventing and treating plant diseases
CN108575999B (en) Application of AHL molecule as chemical pesticide sterilization synergist in plant disease control caused by Burkholderia cepacia
CN113142233B (en) Compatibility research of matrine and Lecanicillium wilsonii and insecticide combined with matrine and Lecanicillium wilsonii
CN114190392B (en) Sterilization composition containing difenoconazole and ketoconazole and application thereof
CN112715577B (en) Application of matrine and beauveria bassiana in preventing and treating common circium japonicum
CN113796392B (en) Fungicide of derris elliptica extract and preparation method and application thereof
CN114128729B (en) Application of chinaberry leaf and fructus evodiae volatile oil in prevention and treatment of phytophthora blight of capsicum

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant